Aryl aniline derivatives as β2 adrenergic receptor agonists

ABSTRACT

The invention provides novel β 2  adrenergic receptor agonist compounds. The invention also provides pharmaceutical compositions comprising such compounds, methods of using such compounds to treat diseases associated with β 2  adrenergic receptor activity, and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 11/033,198filed Jan. 11, 2005 now U.S. Pat. No. 7,622,467 which claims the benefitof U.S. Provisional Application No. 60/535,784, filed on Jan. 12, 2004,the disclosures of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention is directed to novel β₂ adrenergic receptor agonists. Theinvention is also directed to pharmaceutical compositions comprisingsuch compounds, methods of using such compounds to treat diseasesassociated with β₂ adrenergic receptor activity, and processes andintermediates useful for preparing such compounds.

BACKGROUND OF THE INVENTION

β₂ adrenergic receptor agonists are recognized as effective drugs forthe treatment of pulmonary diseases such as asthma and chronicobstructive pulmonary disease (including chronic bronchitis andemphysema). β₂ adrenergic receptor agonists are also useful for treatingpre-term labor, and are potentially useful for treating neurologicaldisorders and cardiac disorders. In spite of the success that has beenachieved with certain β₂ adrenergic receptor agonists, current agentspossess less than desirable duration of action, potency, selectivity,and/or onset. Thus, there is a need for additional β₂ adrenergicreceptor agonists having improved properties, such as improved durationof action, potency, selectivity, and/or onset.

SUMMARY OF THE INVENTION

The invention provides novel compounds that possess β₂ adrenergicreceptor agonist activity. Among other properties, compounds of theinvention have been found to be potent and selective β₂ adrenergicreceptor agonists. In addition, compounds of the invention have beenfound to possess a surprising and unexpectedly long duration of action,which is expected to allow for once-daily, or even less frequent,dosing.

Accordingly, this invention provides a compound of formula (I):

wherein:

each of R¹, R², R³, and R⁴ is independently selected from hydrogen,hydroxy, amino, halo, —CH₂OH and —NHCHO, or R¹ and R² taken together areselected from —NHC(═O)CH═CH—, —CH═CHC(═O)NH—, —NHC(═O)S—; and—SC(═O)NH—;

one of R⁵ and R⁶ is —[X—C₁₋₆alkylenyl]_(n)-NR¹⁰R¹¹ orC₁₋₆alkylenyl-NR¹²R¹³,

and the other of R⁵ and R⁶ is selected from hydrogen, hydroxy,C₁₋₄alkoxy, and C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substitutedwith halo,

wherein

-   -   each X is independently selected from —O—, —NH—, —S—, —NHSO₂—,        —SO₂NH—, —NHC(═O)—, and —C(═O)NH—;    -   each of R¹⁰, R¹¹, R¹², and R¹³ is independently hydrogen or        C₁₋₄alkyl; or    -   R¹⁰ and R¹¹, together with the nitrogen atom to which they are        attached, or R¹⁰, together with the nitrogen atom to which it is        attached and a carbon atom of the adjacent C₁₋₆alkylenyl, or R¹²        and R¹³, together with the nitrogen atom to which they are        attached, or R¹², together with the nitrogen atom to which it is        attached and a carbon atom of the adjacent C₁₋₆alkylenyl, form a        heterocyclic or heteroaryl ring having from 5 to 7 ring atoms,        and optionally containing an additional heteroatom selected from        oxygen, nitrogen, and sulfur, wherein nitrogen is optionally        substituted with —S(O)₂—C₁₋₄alkyl; and    -   n is 1, 2, or 3; and

each of R⁷, R⁸, and R⁹ is independently hydrogen or C₁₋₆alkyl;

or a pharmaceutically-acceptable salt or solvate or stereoisomerthereof.

The invention also provides pharmaceutical compositions comprising acompound of the invention and a pharmaceutically-acceptable carrier. Theinvention further provides combinations comprising a compound of theinvention and one or more other therapeutic agents and pharmaceuticalcompositions comprising such combinations.

The invention also provides a method of treating a mammal having adisease or condition associated with β₂ adrenergic receptor activity(e.g. a pulmonary disease, such as asthma or chronic obstructivepulmonary disease, pre-term labor, a neurological disorder, a cardiacdisorder, or inflammation), the method comprising administering to themammal, a therapeutically effective amount of a compound of theinvention. The invention further provides a method of treatmentcomprising administering a therapeutically effective amount of acombination of a compound of the invention and one or more othertherapeutic agents.

The invention also provides a method of treating a mammal having adisease or condition associated with β₂ adrenergic receptor activity,the method comprising administering to the mammal, a therapeuticallyeffective amount of a pharmaceutical composition of the invention.

The compounds of the invention can also be used as research tools, i.e.to study biological systems or samples, or for studying the activity ofother chemical compounds. Accordingly, in another of its method aspects,the invention provides a method of using a compound of formula (I), or apharmaceutically acceptable salt or solvate or stereoisomer thereof, asa research tool for studying a biological system or sample or fordiscovering new β₂ adrenergic receptor agonists.

In separate and distinct aspects, the invention also provides syntheticprocesses and intermediates described herein, which are useful forpreparing compounds of the invention.

The invention also provides a compound of the invention as describedherein for use in medical therapy, as well as the use of a compound ofthe invention in the manufacture of a formulation or medicament fortreating a mammal having a disease or condition associated with β₂adrenergic receptor activity (e.g. a pulmonary disease, such as asthmaor chronic obstructive pulmonary disease, pre-term labor, a neurologicaldisorder, a cardiac disorder, or inflammation).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an x-ray powder diffraction pattern of the product of Example17b.

FIG. 2 is a differential scanning calorimetry trace of the product ofExample 17b.

FIG. 3 is an x-ray powder diffraction pattern of the product of Example17c.

FIG. 4 is a differential scanning calorimetry trace of the product ofExample 17c.

FIG. 5 is an x-ray powder diffraction pattern of the product of Example17d.

FIG. 6 is a differential scanning calorimetry trace of the product ofExample 17d.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel aryl aniline β₂ adrenergic receptoragonists of formula (I), or pharmaceutically-acceptable salts orsolvates or stereoisomers thereof. The following substituents and valuesare intended to provide representative examples of various aspects ofthe invention. These representative values are intended to furtherdefine such aspects and are not intended to exclude other values orlimit the scope of the invention.

In a specific aspect of the invention, R¹ is halo, —CH₂OH, or —NHCHO.

In other specific aspects, R¹ is chloro, —CH₂OH, or —NHCHO; or R¹ is—CH₂OH or —NHCHO.

In a specific aspect, R² is hydrogen.

In a specific aspect, R³ is hydroxy or amino.

In specific aspects, R⁴ is hydrogen or halo; or R⁴ is hydrogen orchloro.

In a specific aspect, R¹ is —NHCHO, R³ is hydroxy, and R² and R⁴ areeach hydrogen.

In another specific aspect, R¹ and R² taken together are —NHC(═O)CH═CH—or —CH═CHC(═O)NH—, R³ is hydroxy, and R⁴ is hydrogen.

In another specific aspect, R¹ is —CH₂OH, R³ is hydroxy, and R² and R⁴are each hydrogen.

In yet another specific aspect, R¹ and R⁴ are chloro, R³ is amino, andR² is hydrogen.

In still another specific aspect, R¹ and R² taken together are—NHC(═O)S— or —SC(═O)NH—, R³ is hydroxy, and R⁴ is hydrogen.

In a specific aspect, R⁵ or R⁶ is —[X—C₁₋₆alkylenyl]-NR¹⁰R¹¹, where n,X, R¹⁰ and R¹¹ are defined as in formula (I).

In another specific aspect, R⁵ or R⁶ is —[O—C₁₋₆allylenyl]_(n)-NR¹⁰R¹¹where R¹⁰ and R¹¹ are defined as in formula (I) and n is 1 or 2.

In another specific aspect, R⁵ or R⁶ is —O—C₁₋₆alkylenyl-NR¹⁰R¹¹ whereeach of R¹⁰ and R¹¹ is independently hydrogen or C₁₋₄alkyl.Representative R⁵ or R⁶ values include, but are not limited to,—O(CH₂)₂NH₂, —O(CH₂)₃N(CH₃)₂, —O(CH₂)₄NH₂, and —OCH₂C(CH₃)₂NH₂.

In another specific aspect, R⁵ or R⁶ is —O—C₁₋₆alkylenyl-NR¹⁰R¹¹, whereR¹⁰ and R¹¹, together with the nitrogen atom to which they are attached,form a piperazinyl ring. For example, R⁵ or R⁶ is—O(CH₂)₂-1-piperazinyl.

In yet another specific aspect, R⁵ or R⁶ is C₁₋₆alkylenyl-NR¹²R¹³ whereR¹² and R¹³ are defined as in formula (I).

In yet another specific aspect, R⁵ or R⁶ is C₁₋₆alkylenyl-NR¹²R¹³ whereeach of R¹² and R¹³ is independently hydrogen or C₁₋₄alkyl.Representative R⁵ or R⁶ values within this aspect, include, but are notlimited to, —(CH₂)₂NH₂, —(CH₂)₂N(CH₃)₂, and —CH₂C(CH₃)₂NH₂.

In still other specific aspects, R⁵ or R⁶ is C₁₋₄alkyl, optionallysubstituted with halo, for example, CF₃; or R⁵ or R⁶ is C₁₋₄alkoxy, forexample, —OCH₃; or R⁵ or R⁶ is hydrogen; or R⁵ or R⁶ is hydroxy.

In a specific aspect, R⁷ is hydrogen.

In a specific aspect, R⁸ is hydrogen.

In a specific aspect, R⁹ is hydrogen.

In one aspect, the invention provides a compound of formula (II):

wherein:

R¹ is —CH₂OH or —NHCHO, and R² is hydrogen; or R¹ and R² taken togetherare —NHC(═O)CH═CH— or —CH═CHC(═O)NH—;

one of R⁵ and R⁶ is —[O—C₁₋₆alkylenyl]_(n)-NR¹⁰R¹¹ orC₁₋₆alkylenyl-NR¹²R¹³,

and the other of R⁵ and R⁶ is selected from hydrogen, hydroxy,C₁₋₄alkoxy, and C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substitutedwith halo,

wherein

-   -   each of R¹⁰, R¹¹, R¹², and R¹³, is independently hydrogen or        C₁₋₄alkyl; or    -   R¹⁰ and R¹¹, together with the nitrogen atom to which they are        attached, or

R¹⁰, together with the nitrogen atom to which it is attached and acarbon atom of the adjacent C₁₋₆alkylenyl, or R¹² and R¹³, together withthe nitrogen atom to which they are attached, or R¹², together with thenitrogen atom to which it is attached and a carbon atom of the adjacentC₁₋₆alkylenyl, form a heterocyclic or heteroaryl ring having from 5 to 7ring atoms and optionally containing an additional heteroatom selectedfrom oxygen, nitrogen, and sulfur, wherein nitrogen is optionallysubstituted with —S(O)₂—C₁₋₄alkyl; and

-   -   n is 1 or 2;

or a pharmaceutically-acceptable salt or solvate or stereoisomerthereof.

In another aspect, the invention provides compounds of formula (II) inwhich R⁵ is —[O—C₁₋₆alkylenyl]_(n)-NR¹⁰R¹¹ or C₁₋₆alkylenyl-NR¹²R¹³ andR⁶ is hydrogen.

In another aspect, the invention provides compounds of formula (II) inwhich R⁵ is C₁₋₄alkoxy and R⁶ is —[O—C₁₋₆alkylenyl]_(n)-NR¹⁰R¹¹ orC₁₋₆alkylenyl-NR¹²R¹³.

In another aspect, the invention provides compounds of formula (II) inwhich

-   -   R⁵ is selected from —O—C₁₋₆alkylenyl-NR¹⁰R¹¹ and        C₁₋₆alkylenyl-NR¹²R¹³ and R⁶ is hydrogen; or    -   R⁵ is C₁₋₄alkoxy and R⁶ is —C₁₋₆alkylenyl-NR¹²R¹³,    -   wherein each of R¹⁰, R¹¹, R¹² and R¹³ is independently hydrogen        or C₁₋₄, or R¹⁰ and R¹¹, together with the nitrogen atom to        which they are attached, form a piperazinyl ring.

A specific group of compounds within this aspect, is the group in whichR¹ and R² taken together are —NHC(═O)CH═CH—or —CH═CHC(═O)NH—.

Another specific group of compounds within this aspect, is the group inwhich R⁵ is —O—C₁₋₆alkylenyl-NR¹⁰R¹¹ and R⁶ is hydrogen.

In still other specific aspects, the invention provides compounds offormula (II) in which the variables R¹, R², R⁵, and R⁶ take the valueslisted in Table I below.

TABLE I Example No. R¹ and R² R⁵ R⁶ 1 —NHC(═O)CH═CH— —OCH₂C(CH₃)₂NH₂ H 2—NHC(═O)CH═CH— —O(CH₂)₂NH₂ H 3 —NHC(═O)CH═CH— —O(CH₂)₃NH₂ H 4—NHC(═O)CH═CH— —O(CH₂)₄NH₂ H 5 —NHC(═O)CH═CH— —O(CH₂)₂O(CH₂)₂NH₂ H 6—NHC(═O)CH═CH— —O(CH₂)₂-4-morpholinyl H 7 —NHC(═O)CH═CH——O(CH₂)₂-2-piperazinyl H 8 —NHC(═O)CH═CH— —OCH₂-1-pyridinyl H 9—NHC(═O)CH═CH— —OCH₂C(CH₃)₂NH₂ CF₃ 10 —NHC(═O)CH═CH——O(CH₂)₂-1,4-piperazinyl-SO₂CH₃ H 11 —NHC(═O)CH═CH— —(CH₂)₂NH₂ H 12—NHC(═O)CH═CH— —(CH₂)₂N(CH₃)₂ H 13 —NHC(═O)CH═CH— —CH₂C(CH₃)₂NH₂ H 14 R¹= —NHCHO R² = H —OCH₂C(CH₃)₂NH₂ H 15 —NHC(═O)CH═CH— —OCH₃ —(CH₂)₂NH₂ 16R¹ = —NHCHO R² = H —OCH₃ —(CH₂)₂NH₂

Particular mention may be made of the following compounds:

-   5-[(R)-2-(2-{4-[4-(2-amino-2-methyl    -propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;-   8-hydroxy-5-[(R)-1-hydroxy-2-(2-{4-[4-(2-piperazin-1-yl-ethoxy)-phenylamino]-phenyl}-ethylamino)-ethyl]-1H-quinolin-2-one;-   5-[(R)-2-(2-{4-[4-(2-amino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;-   5-[(R)-2-(2-{4-[4(2-dimethylamino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin    -2-one;-   5-[(R)-2-(2-{4-[3-(2-amino-ethyl)-4-methoxy    -phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H    -quinolin-2-one; where the chemical nomenclature conforms to that of    the automatic naming program AutoNom, as provided by MDL Information    Systems, GmbH (Frankfurt, Germany).

As illustrated above, the compounds of the invention contain one or morechiral centers. Accordingly, the invention includes racemic mixtures,pure stereoisomers (i.e. individual enantiomers or diastereomers), andstereoisomer-enriched mixtures of such isomers, unless otherwiseindicated. When a particular stereoisomer is shown, it will beunderstood by those skilled in the art, that minor amounts of otherstereoisomers may be present in the compositions of this inventionunless otherwise indicated, provided that the utility of the compositionas a whole is not eliminated by the presence of such other isomers.

In particular, compounds of the invention contain a chiral center at thealkylene carbon in formulas (I) and (II) to which the hydroxy group isattached. When a mixture of stereoisomers is employed, it isadvantageous for the amount of the stereoisomer with the (R) orientationat the chiral center bearing the hydroxy group to be greater than theamount of the corresponding (S) stereoisomer. When comparingstereoisomers of the same compound, the (R) stereoisomer is preferredover the (S) stereoisomer.

Definitions

When describing the compounds, compositions and methods of theinvention, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkyl groups include, by way of example, methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

The term “alkoxy” means a monovalent group —O-alkyl, where alkyl isdefined as above. Representative alkoxy groups include, by way ofexample, methoxy, ethoxy, propoxy, butoxy, and the like.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupcontaining at least one carbon-carbon double bond, typically 1 or 2carbon-carbon double bonds, and which may be linear or branched orcombinations thereof. Unless otherwise defined, such alkenyl groupstypically contain from 2 to 10 carbon atoms. Representative alkenylgroups include, by way of example, vinyl, allyl, isopropenyl,but-2-enyl, n-pent-2-enyl, n-hex-2-enyl, n-hept-2-enyl, n-oct-2-enyl,n-non-2-enyl, n-dec-4-enyl, n-dec-2,4-dienyl and the like.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupcontaining at least one carbon-carbon triple bond, typically 1carbon-carbon triple bond, and which may be linear or branched orcombinations thereof. Unless otherwise defined, such alkynyl groupstypically contain from 2 to 10 carbon atoms. Representative alkynylgroups include, by way of example, ethynyl, propargyl, but-2-ynyl andthe like.

The term “alkylenyl” means a divalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkylenyl groups typically contain from 1 to 10 carbonatoms. Representative alkylenyl groups include, by way of example,methylene, ethylene, n-propylene, n-butylene, propane-1,2-diyl(1-methylethylene), 2-methylpropane-1,2-diyl (1,1-dimethylethylene) andthe like.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen,oxygen, and sulfur. Unless otherwise defined, such heteroaryl groupstypically contain from 5 to 10 total ring atoms. Representativeheteroaryl groups include, by way of example, pyrroyl, isoxazolyl,isothiazolyl, pyrazolyl, pyridyl (or, equivalently, pyridinyl),oxazolyl, oxadiazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl,tetrazolyl, furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl,pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl,benzothiophenyl, quinolyl, indolyl, isoquinolyl and the like, where thepoint of attachment is at any available carbon or nitrogen ring atom.

The term “heterocyclyl” or “heterocyclic ring” means a monovalentsaturated or partially unsaturated cyclic non-aromatic group, which maybe monocyclic or multicyclic (i.e., fused or bridged), and whichcontains at least one heteroatom (typically 1 to 3 heteroatoms) selectedfrom nitrogen, oxygen, and sulfur. Unless otherwise defined, suchheterocyclyl groups typically contain from 5 to 10 total ring atoms.Representative heterocyclyl groups include, by way of example,pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, morpholinyl,indolin-3-yl, 2-imidazolinyl, 1,2,3,4-tetrahydroisoquinolin-2-yl,quinuclidinyl, and the like.

The term “halo” means fluoro, chloro, bromo or iodo.

The term “treatment” as used herein means the treatment of a disease ormedical condition in a patient, such as a mammal (particularly a human)which includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The phrase “disease or condition associated with β₂ adrenergic receptoractivity” includes all medical conditions alleviated by treatment withβ₂ adrenergic receptor agonist and includes all disease states and/orconditions that are acknowledged now, or that are found in the future,to be associated with β₂ adrenergic receptor activity. Such diseasestates include, but are not limited to, pulmonary diseases, such asasthma and chronic obstructive pulmonary disease (including chronicbronchitis and emphysema), as well as neurological disorders and cardiacdisorders. β₂ adrenergic receptor activity is also known to beassociated with pre-term labor (see U.S. Pat. No. 5,872,126) and sometypes of inflammation (see International Patent Application PublicationNumber WO 99/30703 and U.S. Pat. No. 5,290,815).

The term “pharmaceutically-acceptable salt” refers to a salt preparedfrom a base or acid which is acceptable for administration to a patient,such as a mammal. Such salts can be derived frompharmaceutically-acceptable inorganic or organic bases and frompharmaceutically-acceptable inorganic or organic acids.

Salts derived from pharmaceutically-acceptable acids include, but arenot limited to, acetic, benzenesulfonic, benzoic, camphosulfonic,citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic, xinafoic (1-hydroxy-2-naphthoic acid),1,5-naphthalene disulfonic, cinnamic, and the like. Salts derived fromfumaric, hydrobromic, hydrochloric, acetic, sulfuric, methanesulfonic,1,5-naphthalene disulfonic, xinafoic, oxalic, tartaric, and4-methyl-cinnamic acids are of particular interest.

Salts derived from pharmaceutically-acceptable inorganic bases includealuminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, manganous, potassium, sodium, zinc and the like.Salts derived from pharmaceutically-acceptable organic bases includesalts of primary, secondary and tertiary amines, including substitutedamines, cyclic amines, naturally-occurring amines and the like, such asarginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of the invention or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include by way of example, water, methanol, ethanol,isopropanol, acetic acid, and the like. When the solvent is water, thesolvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically-acceptablesalt or solvate of stereoisomer thereof” is intended to include allpermutations of salts, solvates and stereoisomers, such as a solvate ofa pharmaceutically-acceptable salt of a stereoisomer of a compound offormula (I).

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; sulfonic ester groups, such as mesylate, tosylate, brosylate,nosylate and the like; and acyloxy groups, such as acetoxy,trifluoroacetoxy and the like.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl; alkoxycarbonylgroups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups,such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);arylmethyl groups, such as benzyl (Bn), trityl (Tr), and1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBS); and the like.

The term “hydroxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a hydroxy group. Representativehydroxy-protecting groups include, but are not limited to, alkyl groups,such as methyl, ethyl, and tert-butyl; acyl groups, for example alkanoylgroups, such as acetyl; arylmethyl groups, such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); silyl groups, such as trimethylsilyl (TMS) andtert-butyldimethylsilyl (TBS); and the like.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods and procedures.Although a particular aspect of the present invention is illustrated inthe schemes below, those skilled in the art will recognize that allaspects of the present invention can be prepared using the methodsdescribed herein or by using other methods, reagents and startingmaterials known to those skilled in the art. It will also be appreciatedthat where typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group, as well assuitable conditions for protection and deprotection, are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

In one method of synthesis, compounds of formulas (I) and (II) areprepared as illustrated in Scheme A. (The substituents and variablesshown in the following schemes have the definitions provided aboveunless otherwise indicated.)

where P¹ represents a hydroxy-protecting group, P² represents ahydroxy-protecting group, and L represents a leaving group, such asbromo.

As shown in Scheme A, a compound of formula 1 is first reacted with anaryl amine (2) to provide an intermediate of formula 3. Typically, thisreaction is conducted in an organic solvent in the presence of base anda transition metal catalyst and arylphosphine ligand with heating. Auseful catalyst for coupling of an aryl group to an aryl amine istris(dibenzylidenacetone)dipalladium(0) together withrac-2,2′-bis(diphenylphosphino)-1,1′-binapthyl. The reaction istypically heated at a temperature of between about 50° C. and about 120°C. for between about 0.25 and about 12 hours. The protecting group P¹ istypically a silyl protecting group, which is typically removed from theintermediate of formula 3 using a fluoride or acid reagent, to providean intermediate of formula 4. The protecting group P² is typically abenzyl protecting group, which is typically removed from theintermediate of formula 4 by hydrogenation using a palladium on carboncatalyst, to provide the product.

An alternative method of preparing intermediate 3 is illustrated inScheme B.

The conditions for the coupling of intermediates 5 and 6 in Scheme B toproduce intermediate 3 are typically the same as those used to coupleintermediates 1 and 2 in Scheme A.

Yet another alternative method of preparing intermediate 3 isillustrated in Scheme C.

The reaction of Scheme C is typically conducted in a polar aproticsolvent in the presence of base. Typical suitable solvents includedimethylsulfoxide, dimethyl formamide, dimethylacetamide and the like.The reaction is typically heated at a temperature of between about 60°C. and about 140° C. for between about 0.25 and about 4 hours.

The compounds of formula 1 and 7 employed in the reactions described inthis application are readily prepared by procedures known in the art,and described, for example, in U.S. Pat. Nos. 6,653,323 B2 and 6,670,376B1, which are incorporated herein by reference, and references therein.Intermediate 5 can be prepared by reaction of intermediate 7 with2-(4-aminophenyl)ethylamine in an aprotic solvent with heating.

Intermediates 2 and 6 are available commercially or are prepared fromreadily available starting materials. For example, when R⁵ is—[O—C₁₋₆alkylenyl]_(n)-NR¹⁰R¹¹ and R⁶ is hydrogen, intermediate 2′, ofgeneral formula 2, can be prepared by the process of Scheme D

where R^(5a) is defined such that —OR^(5a) is—[O—C₁₋₆alkylenyl]_(n)-NR¹⁰R¹¹. As one example of suitable reactionconditions for Scheme D, the reaction is conducted in dimethylsulfoxidein the presence of sodium hydride.

When, for example, R⁵ is C₁₋₆alkylenyl-NR¹²R¹³ and R⁶ is hydrogen,intermediate 2″, of general formula 2, can be prepared by the process ofScheme E

where R^(5b) is C₁₋₆alkylenyl.

An intermediate of formula 8 can be prepared by reacting an intermediateof formula 2 with a phenethylamine substituted with a leaving group atthe 4-position of the phenyl ring, for example, 4-bromophenethylamine.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediate thereto are described in the Examples below.

Accordingly, in a method aspect, the invention provides a process forpreparing a compound of formula (I), or a salt or stereoisomer orprotected derivative thereof, the process comprising:

(a) reacting

-   -   (i) a compound of formula (III):

-   -    with a compound of formula (IV):

-   -    in the presence of a transition metal catalyst; or    -   (ii) a compound of formula (V):

-   -    with a compound of formula (VI):

-   -    wherein P¹ is a hydroxy-protecting group, each of R^(1a),        R^(2a), R^(3a), and R^(4a) is independently either defined to be        the same as R¹, R², R³, and R⁴ in formula (I) or is —OP²,        wherein P² is a hydroxy-protecting group; one of A and B is a        leaving group and the other of A and B is —NH₂; L is a leaving        group; and R⁵, R⁶, R⁷, R⁸, and R⁹ are defined as in formula (I),        to provide a compound of formula (VII);

(b) removing the protecting group P¹ to provide a compound of formula(VIII):

(c) when any of R^(1a), R^(2a), R^(3a), or R^(4a) is —OP², removing theprotecting group P² to provide a compound of formula (I), or a salt orstereoisomer thereof.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising acompound of the invention. Accordingly, the compound, preferably in theform of a pharmaceutically-acceptable salt, can be formulated for anysuitable form of administration, such as oral or parenteraladministration, or administration by inhalation.

By way of illustration, the compound can be admixed with conventionalpharmaceutical carriers and excipients and used in the form of powders,tablets, capsules, elixirs, suspensions, syrups, wafers, and the like.Such pharmaceutical compositions will contain from about 0.05 to about90% by weight of the active compound, and more generally from about 0.1to about 30%. The pharmaceutical compositions may contain commoncarriers and excipients, such as cornstarch or gelatin, lactose,magnesium sulfate, magnesium stearate, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, andalginic acid. Disintegrators commonly used in the formulations of thisinvention include croscarrnellose, microcrystalline cellulose,cornstarch, sodium starch glycolate and alginic acid.

A liquid composition will generally consist of a suspension or solutionof the compound or pharmaceutically-acceptable salt in a suitable liquidcarrier(s), for example ethanol, glycerine, sorbitol, non-aqueoussolvent such as polyethylene glycol, oils or water, optionally with asuspending agent, a solubilizing agent (such as a cyclodextrin),preservative, surfactant, wetting agent, flavoring or coloring agent.Alternatively, a liquid formulation can be prepared from areconstitutable powder.

For example a powder containing active compound, suspending agent,sucrose and a sweetener can be reconstituted with water to form asuspension; a syrup can be prepared from a powder containing activeingredient, sucrose and a sweetener.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidcompositions. Examples of such carriers include magnesium stearate,starch, lactose, sucrose, microcrystalline cellulose and binders, forexample polyvinylpyrrolidone. The tablet can also be provided with acolor film coating, or color included as part of the carrier(s). Inaddition, active compound can be formulated in a controlled releasedosage form as a tablet comprising a hydrophilic or hydrophobic matrix.

A composition in the form of a capsule can be prepared using routineencapsulation procedures, for example by incorporation of activecompound and excipients into a hard gelatin capsule. Alternatively, asemi-solid matrix of active compound and high molecular weightpolyethylene glycol can be prepared and filled into a hard gelatincapsule; or a solution of active compound in polyethylene glycol or asuspension in edible oil, for example liquid paraffin or fractionatedcoconut oil can be prepared and filled into a soft gelatin capsule.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, poly-vinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose. Lubricants that canbe used include magnesium stearate or other metallic stearates, stearicacid, silicone fluid, talc, waxes, oils and colloidal silica.

Flavoring agents such as peppermint, oil of wintergreen, cherryflavoring or the like can also be used. Additionally, it may bedesirable to add a coloring agent to make the dosage form moreattractive in appearance or to help identify the product.

The compounds of the invention and their pharmaceutically-acceptablesalts that are active when given parenterally can be formulated forintramuscular, intrathecal, or intravenous administration.

A typical composition for intra-muscular or intrathecal administrationwill consist of a suspension or solution of active ingredient in an oil,for example arachis oil or sesame oil. A typical composition forintravenous or intrathecal administration will consist of a sterileisotonic aqueous solution containing, for example active ingredient anddextrose or sodium chloride, or a mixture of dextrose and sodiumchloride. Other examples are lactated Ringer's injection, lactatedRinger's plus dextrose injection, Normosol-M and dextrose, Isolyte E,acylated Ringer's injection, and the like. Optionally, a co-solvent, forexample, polyethylene glycol; a chelating agent, for example,ethylenediamine tetraacetic acid; a solubilizing agent, for example, acyclodextrin; and an anti-oxidant, for example, sodium metabisulphite,may be included in the formulation. Alternatively, the solution can befreeze dried and then reconstituted with a suitable solvent just priorto administration.

The compounds of this invention and their pharmaceutically-acceptablesalts which are active on topical administration can be formulated astransdermal compositions or transdermal delivery devices (“patches”).Such compositions include, for example, a backing, active compoundreservoir, a control membrane, liner and contact adhesive. Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, forexample, U.S. Pat. No. 5,023,252. Such patches may be constructed forcontinuous, pulsatile, or on demand delivery of pharmaceutical agents.

One preferred manner for administering a compound of the invention isinhalation. Inhalation is an effective means for delivering an agentdirectly to the respiratory tract. There are three general types ofpharmaceutical inhalation devices: nebulizer inhalers, dry powderinhalers (DPI), and metered-dose inhalers (MDI). Conventional nebulizerdevices produce a stream of high velocity air that causes a therapeuticagent to spray as a mist which is carried into the patient's respiratorytract. The therapeutic agent is formulated in a liquid form such as asolution or a suspension of micronized particles of respirable size,where micronized is typically defined as having about 90% or more of theparticles with a diameter of less than about 10 μm.

A typical formulation for use in a conventional nebulizer device is anisotonic aqueous solution of a pharmaceutical salt of the active agentat a concentration of the active agent of between about 0.05 μg/mL andabout 1 mg/mL. Suitable nebulizer devices are provided commercially, forexample, by PARI GmbH (Starnberg, Germany). Other nebulizer devices havebeen disclosed, for example, in U.S. Pat. No. 6,123,068.

DPI's typically administer a therapeutic agent in the form of a freeflowing powder that can be dispersed in a patient's air-stream duringinspiration. Alternative DPI devices which use an external energy sourceto disperse the powder are also being developed. In order to achieve afree flowing powder, the therapeutic agent can be formulated with asuitable excipient (e.g., lactose or starch). A dry powder formulationcan be made, for example, by combining dry lactose particles withmicronized particles of a suitable form, typically apharmaceutically-acceptable salt, of a compound of the invention (i.e.the active agent) and dry blending. Alternatively, the agent can beformulated without excipients. The formulation is loaded into a drypowder dispenser, or into inhalation cartridges or capsules for use witha dry powder delivery device.

Examples of DPI delivery devices provided commercially include Diskhaler(GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g., U.S. Pat.No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No.6,378,519; Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S.Pat. No. 4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g., U.S.Pat. No. 4,353,365). Further examples of suitable DPI devices aredescribed in U.S. Pat. Nos. 5,415,162, 5,239,993, and 5,715,810 andreferences therein.

MDI's typically discharge a measured amount of therapeutic agent usingcompressed propellant gas. Formulations for MDI administration include asolution or suspension of active ingredient in a liquefied propellant.While chlorofluorocarbons, such as CCl₃F, conventionally have been usedas propellants, due to concerns regarding adverse affects of such agentson the ozone layer, formulations using hydrofluoroalklanes (HFA), suchas 1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3,-heptafluoro-n-propane, (HFA 227) have been developed.Additional components of HFA formulations for MDI administration includeco-solvents, such as ethanol or pentane, and surfactants, such assorbitan trioleate, oleic acid, lecithin, and glycerin. (See, forexample, U.S. Pat. No. 5,225,183, EP 0717987 A2, and WO 92/22286.)

Thus, a suitable formulation for MDI administration can include fromabout 0.001% to about 2% by weight of the present crystalline form, fromabout 0% to about 20% by weight ethanol, and from about 0% to about 5%by weight surfactant, with the remainder being the FIFA propellant. Inone approach, to prepare the formulation, chilled or pressurizedhydrofluoroalkane is added to a vial containing the present crystallineform, ethanol (if present) and the surfactant (if present). To prepare asuspension, the pharmaceutical salt is provided as micronized particles.The formulation is loaded into an aerosol canister, which forms aportion of an MDI device. Examples of MDI devices developed specificallyfor use with HFA propellants are provided in U.S. Pat. Nos. 6,006,745and 6,143,227.

In an alternative preparation, a suspension formulation is prepared byspray drying a coating of surfactant on micronized particles of apharmaceutical salt of active compound. (See, for example, WO 99/53901and WO 00/61108.) For additional examples of processes of preparingrespirable particles, and formulations and devices suitable forinhalation dosing see U.S. Pat. Nos. 6,268,533, 5,983,956, 5,874,063,and 6,221,398, and WO 99/55319 and WO 00/30614.

It will be understood that any form of the compounds of the invention,(i.e. free base, pharmaceutical salt, or solvate) that is suitable forthe particular mode of administration, can be used in the pharmaceuticalcompositions discussed above.

The active compounds are expected to be effective over a wide dosagerange and to be administered in a therapeutically effective amount. Itwill be understood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

A compound can be administered in a periodic dose: weekly, multipletimes per week, daily, or multiple doses per day. The treatment regimenmay require administration over extended periods of time, for example,for several weeks or months, or the treatment regimen may requirechronic administration. Suitable doses for oral administration are inthe general range of from about 0.05 μg/day to about 100 mg/day,preferably 0.5 to 1000 μg/day.

Suitable doses of the therapeutic agents for inhalation administrationare in the general range of from about 0.05 μg/day to about 1000 μg/day,preferably from about 0.1 μg/day to about 500 μg/day. It will beunderstood that the fraction of active agent delivered to the lungcharacteristic of particular delivery devices is taken into account indetermining suitable doses for inhalation administration.

Among other properties, compounds of the invention have been found to bepotent and selective agonists of the β₂ adrenergic receptor. Inparticular, compounds of the invention demonstrate excellent selectivityfor the β₂ adrenergic receptor as compared with the β₁ and β₃ adrenergicreceptors. Furthermore, compounds of the invention have been found topossess surprising and unexpected duration of action. As described inthe biological assays below, compounds of the invention demonstratedduration of action greater than 24 hours in an animal model ofbronchoprotection.

The invention thus provides a method of treating a disease or conditionin a mammal associated with β₂ adrenergic receptor activity, the methodcomprising administering to the mammal a therapeutically effectiveamount of a compound of the invention or of a pharmaceutical compositioncomprising a compound of the invention.

The present active agents can also used as part of a combinationcomprising, in addition, one or more other therapeutic agents. Forexample, the present agents can be administered together with one ormore therapeutic agents selected from anti-inflammatory agents (e.g.corticosteroids and non-steroidal anti-inflammatory agents (NSAIDs),antichlolinergic agents (particularly muscarinic receptor antagonists),other β₂ adrenergic receptor agonists, antiinfective agents (e.g.antibiotics or antivirals) or antihistamines. The invention thusprovides, in a further aspect, a combination comprising a compound ofthe invention and one or more therapeutic agents, for example, ananti-inflammatory agent, an antichlolinergic agent, another β₂adrenergic receptor agonist, an antiinfective agent or an antihistamine.

The other therapeutic agents can be used in the form ofpharmaceutically-acceptable salts or solvates. As appropriate, the othertherapeutic agents can be used as optically pure stereoisomers.

Suitable anti-inflammatory agents include corticosteroids and NSAIDs.Suitable corticosteroids which may be used in combination with thecompounds of the invention are those oral and inhaled corticosteroidsand their pro-drugs which have anti-inflammatory activity. Examplesinclude methyl prednisolone, prednisolone, dexamethasone, fluticasonepropionate,6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16αmethyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydro-furan-3S-yl) ester, beclomethasone esters (e.g.the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g. the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,and ST-126. Preferred corticosteroids include fluticasone propionate,6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester and6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester, more preferably6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester.

Suitable NSAIDs include sodium cromoglycate; nedocromil sodium;phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4 inhibitorsor mixed PDE3/PDE4 inhibitors); leukotriene antagonists (e.g.monteleukast); inhibitors of leukotriene synthesis; iNOS inhibitors;protease inhibitors, such as tryptase and elastase inhibitors; beta-2integrin antagonists and adenosine receptor agonists or antagonists(e.g. adenosine 2a agonists); cytokine antagonists (e.g. chemokineantagonists such as, an interleukin antibody (αIL antibody),specifically, an αIL-4 therapy, an αIL-13 therapy, or a combinationthereof); or inhibitors of cytokine synthesis. Suitable otherβ₂-adrenoreceptor agonists include salmeterol (e.g. as the xinafoate),salbutamol (e.g. as the sulphate or the free base), formoterol (e.g. asthe fumarate), fenoterol or terbutaline and salts thereof.

Also of interest is use of the present active agent in combination witha phosphodiesterase 4 (PDE4) inhibitor or a mixed PDE3/PDE4 inhibitor.Representative phosphodiesterase-4 (PDE4) inhibitors or mixed PDE3/PDE4inhibitors include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically-acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

Suitable anticholinergic agents are those compounds that act asantagonists at the muscarinic receptor, in particular those compoundswhich are antagonists of the M₁, M₂, or M₃ receptors, or of combinationsthereof. Exemplary compounds include the alkaloids of the belladonnaplants as illustrated by the likes of atropine, scopolamine,homatropine, hyoscyamine; these compounds are normally administered as asalt, being tertiary amines. These drugs, particularly the salt forms,are readily available from a number of commercial sources or can be madeor prepared from literature data via, to wit:

Atropine—CAS-51-55-8 or CAS-51-48-1 (anhydrous form), atropinesulfate—CAS-5908-99-6; atropine oxide—CAS-4438-22-6 or its HClsalt—CAS-4574-60-1 and methylatropine nitrate—CAS-52-88-0.

Homatropine—CAS-87-00-3, hydrobromide salt—CAS-51-56-9, methylbromidesalt—CAS-80-49-9.

Hyoscyamine (d, l)—CAS-101-31-5, hydrobromide salt—CAS-306-03-6 andsulfate salt—CAS-6835-16-1.

Scopolamine—CAS-51-34-3, hydrobromide salt—CAS-6533-68-2, methylbromidesalt—CAS-155-41-9.

Preferred anticholinergics include ipratropium (e.g. as the bromide),sold under the name Atrovent, oxitropium (e.g. as the bromide) andtiotropium (e.g. as the bromide) (CAS-139404-48-1). Also of interestare: methantheline (CAS-53-46-3), propantheline bromide (CAS-50-34-9),anisotropine methyl bromide or Valpin 50 (CAS-80-50-2), clidiniumbromide (Quarzan, CAS-3485-62-9), copyrrolate (Robinul), isopropamideiodide (CAS-71-81-8), mepenzolate bromide (U.S. Pat. No. 2,918,408),tridihexethyl chloride (Pathilone, CAS-4310-35-4), and hexocycliummethylsulfate (Tral, CAS-115-63-9). See also cyclopentolatehydrochloride (CAS-5870-29-1), tropicamide (CAS-1508-75-4),trihexyphenidyl hydrochloride (CAS-144-11-6), pirenzepine(CAS-29868-97-1), telenzepine (CAS-80880-90-9), AF-DX 116, ormethoctramine, and the compounds disclosed in WO01/04118, the disclosureof which is hereby incorporated by reference.

Suitable antihistamines (also referred to as H₁-receptor antagonists)include any one or more of the numerous antagonists known which inhibitH₁-receptors, and are safe for human use. All are reversible,competitive inhibitors of the interaction of histamine withH₁-receptors. The majority of these inhibitors, mostly first generationantagonists, are characterized, based on their core structures, asethanolamines, ethylenediamines, and alkylamines. In addition, otherfirst generation antihistamines include those which can be characterizedas based on piperizine and phenothiazines. Second generationantagonists, which are non-sedating, have a similar structure-activityrelationship in that they retain the core ethylene group (thealkylamines) or mimic a tertiary amine group with piperizine orpiperidine. Exemplary antagonists are as follows:

Ethanolamines: carbinoxamine maleate, clemastine fumarate,diphenylhydramine hydrochloride, and dimenhydrinate.

Ethylenediamines: pyrilamine amleate, tripelennamine HCl, andtripelennamine citrate.

Alkylamines: chlorpheniramine and its salts such as the maleate salt,and acrivastine.

Piperazines: hydroxyzine HCl, hydroxyzine pamoate, cyclizine HCl,cyclizine lactate, meclizine HCl, and cetirizine HCl.

Piperidines: Astemizole, levocabastine HCl, loratadine or itsdescarboethoxy analogue, and terfenadine and fexofenadine hydrochlorideor another pharmaceutically-acceptable salt.

Azelastine hydrochloride is yet another H₁ receptor antagonist which maybe used in combination with a compound of the invention.

Examples of preferred anti-histamines include methapyrilene andloratadine.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof and a corticosteroid. Inparticular, the invention provides a combination wherein thecorticosteroid is fluticasone propionate or wherein the corticosteroidis6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16αmethyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydro-furan-3S-yl) ester.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof and a PDE4 inhibitor.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof and an anticholinergic agent.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof and an antihistamine.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof together with a PDE4 inhibitorand a corticosteroid.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof together with an anticholinergicagent and a corticosteroid.

As used in the above combinations, the term, “a compound of formula (I)”includes a compound of formula (II) and preferred groups thereof, andany individually disclosed compound or compounds.

Accordingly, the pharmaceutical compositions of the invention canoptionally comprise combinations of a compound of formula (I) or apharmaceutically-acceptable salt or solvate or stereoisomer thereof withone or more other therapeutic agents, as described above.

The individual compounds of the combinations of the invention may beformulated separately or formulated together in a single pharmaceuticalcomposition. The individual compounds may be administered eithersequentially or simultaneously in separate or combined pharmaceuticalformulations. Appropriate doses of known therapeutic agents will bereadily appreciated by those skilled in the art. Methods of treatment ofthe invention, therefore, include administration of the individualcompounds of such combinations either sequentially or simultaneously inseparate or combined pharmaceutical formulations.

Thus, according to a further aspect, the invention provides a method oftreating a disease or condition associated with β₂ adrenergic receptoractivity in a mammal, the method comprising administering to the mammala therapeutically effective amount of a combination of a compound offormula (I) or a pharmaceutically-acceptable salt or solvate orstereoisomer thereof and one or more other therapeutic agents.

Since compounds of the invention are β₂ adrenergic receptor agonists,such compounds are also useful as research tools for investigating orstudying biological systems or samples having β₂ adrenergic receptors,or for discovering new β₂ adrenergic receptor agonists. Moreover, sincecompounds of the invention exhibit selectivity for β₂ adrenergicreceptors as compared with binding and functional activity at receptorsof other β adrenergic subtypes, such compounds are also useful forstudying the effects of selective agonism of β₂ adrenergic receptors ina biological system or sample. Any suitable biological system or samplehaving β₂ adrenergic receptors may be employed in such studies which maybe conducted either in vitro or in vivo. Representative biologicalsystems or samples suitable for such studies include, but are notlimited to, cells, cellular extracts, plasma membranes, tissue samples,mammals (such as mice, rats, guinea pigs, rabbits, dogs, pigs, etc.) andthe like.

A biological system or sample comprising a β₂ adrenergic receptor iscontacted with a β₂ adrenergic receptor-agonizing amount of a compoundof the invention. The effects of agonizing the β₂ adrenergic receptorare determined using conventional procedures and equipment, such asradioligand binding assays and functional assays, for example the assayfor ligand-mediated changes in intracellular cyclic adenosinemonophosphate (cAMP) described below, or assays of a similar nature. Aβ₂ adrenergic receptor-agonizing amount of a compound of the inventionwill typically range from about 1 nanomolar to about 1000 nanomolar.When compounds of the invention are used as research tools fordiscovering new β₂ adrenergic receptor agonists, the invention alsoincludes, as separate embodiments, both the generation of comparisondata (using the appropriate assays) and the analysis of the test data toidentify test compounds of interest.

The following non-limiting examples illustrate representativepharmaceutical compositions of the invention. Additional suitablecarriers for formulations of the active compounds of the presentinvention can be found in Remington: The Science and Practice ofPharmacy, 20th Edition, Lippincott Williams & Wilkins, Philadelphia,Pa., 2000.

Formulation Example A

This example illustrates the preparation of a representativepharmaceutical composition for oral administration of a compound of thisinvention:

Ingredients Quantity per tablet, (mg) Active Compound 1 Lactose,spray-dried 148 Magnesium stearate 2

The above ingredients are mixed and introduced into a hard-shell gelatincapsule.

Formulation Example B

This example illustrates the preparation of another representativepharmaceutical composition for oral administration of a compound of thisinvention:

Ingredients Quantity per tablet, (mg) Active Compound 1 Cornstarch 50Lactose 145 Magnesium stearate 5

The above ingredients are mixed intimately and pressed into singlescored tablets.

Formulation Example C

This example illustrates the preparation of a representativepharmaceutical composition for oral administration of a compound of thisinvention.

An oral suspension is prepared having the following composition.

Ingredients Active Compound 3 mg Fumaric acid 0.5 g Sodium chloride 2.0g Methyl paraben 0.1 g Granulated sugar 25.5 g Sorbitol (70% solution)12.85 g Veegum K (Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5mg Distilled water q.s. to 100 mL

Formulation Example D

This example illustrates the preparation of a representativepharmaceutical composition containing a compound of this invention.

An injectable preparation buffered to a pH of 4 is prepared having thefollowing composition:

Ingredients Active Compound 0.1 mg Sodium Acetate Buffer Solution (0.4M) 2.0 mL HCl (1N) q.s. to pH 4 Water (distilled, sterile) q.s. to 20 mL

Formulation Example E

This example illustrates the preparation of a representativepharmaceutical composition for injection of a compound of thisinvention.

A reconstituted solution is prepared by adding 20 mL of sterile water to1 mg of the compound of this invention. Before use, the solution is thendiluted with 200 mL of an intravenous fluid that is compatible with theactive compound. Such fluids are chosen from 5% dextrose solution, 0.9%sodium chloride, or a mixture of 5% dextrose and 0.9% sodium chloride.Other examples are lactated Ringer's injection, lactated Ringer's plus5% dextrose injection, Normosol-M and 5% dextrose, Isolyte E, andacylated Ringer's injection.

Formulation Example F

This example illustrates the preparation of a representativepharmaceutical composition for topical application of a compound of thisinvention.

Ingredients grams Active compound 0.2-10 Span 60 2 Tween 60 2 Mineraloil 5 Petrolatum 10 Methyl paraben 0.15 Propyl paraben 0.05 BHA(butylated hydroxy anisole) 0.01 Water q.s. to 100

All of the above ingredients, except water, are combined and heated to60° C. with stirring. A sufficient quantity of water at 60° C. is thenadded with vigorous stirring to emulsify the ingredients, and water thenadded q.s. 100 g.

Formulation Example G

This example illustrates the preparation of a representativepharmaceutical composition containing a compound of the invention.

An aqueous aerosol formulation for use in a nebulizer is prepared bydissolving 0.1 mg of a pharmaceutical salt of active compound in a 0.9%sodium chloride solution acidified with citric acid. The mixture isstirred and sonicated until the active salt is dissolved. The pH of thesolution is adjusted to a value in the range of from 3 to 8 by the slowaddition of NaOH.

Formulation Example H

This example illustrates the preparation of a dry powder formulationcontaining a compound of the invention for use in inhalation cartridges.

Gelatin inhalation cartridges are filled with a pharmaceuticalcomposition having the following ingredients:

Ingredients mg/cartridge Pharmaceutical salt of active compound 0.2Lactose 25

The pharmaceutical salt of active compound is micronized prior toblending with lactose. The contents of the cartridges are administeredusing a powder inhaler.

Formulation Example I

This example illustrates the preparation of a dry powder formulationcontaining a compound of the invention for use in a dry powderinhalation device.

A pharmaceutical composition is prepared having a bulk formulation ratioof micronized pharmaceutical salt to lactose of 1:200. The compositionis packed into a dry powder inhalation device capable of deliveringbetween about 10 μg and about 100 μg of active drug ingredient per dose.

Formulation Example J

This example illustrates the preparation of a formulation containing acompound of the invention for use in a metered dose inhaler.

A suspension containing 5% pharmaceutical salt of active compound, 0.5%lecithin, and 0.5% trehalose is prepared by dispersing 5 g of activecompound as micronized particles with mean size less than 10 μm in acolloidal solution formed from 0.5 g of trehalose and 0.5 g of lecithindissolved in 100 mL of demineralized water. The suspension is spraydried and the resulting material is micronized to particles having amean diameter less than 1.5 μm. The particles are loaded into canisterswith pressurized 1,1,1,2-tetrafluoroethane.

Formulation Example K

This example illustrates the preparation of a formulation containing acompound of the invention for use in a metered dose inhaler.

A suspension containing 5% pharmaceutical salt of active compound and0.1% lecithin is prepared by dispersing 10 g of active compound asmicronized particles with mean size less than 10 μm in a solution formedfrom 0.2 g of lecithin dissolved in 200 mL of demineralized water. Thesuspension is spray dried and the resulting material is micronized toparticles having a mean diameter less than 1.5 μm. The particles areloaded into canisters with pressurized1,1,1,2,3,3,3-heptafluoro-n-propane.

Biological Assays

The compounds of this invention, and their pharmaceutically-acceptablesalts, exhibit biological activity and are useful for medical treatment.The ability of a compound to bind to the β₂ adrenergic receptor, as wellas its selectivity, agonist potency, and intrinsic activity can bedemonstrated using Tests A-B below, or can be demonstrated using othertests that are known in the art.

Abbreviations

-   -   % Eff % efficacy    -   ATCC American Type Culture Collection    -   BSA Bovine Serum Albumin    -   cAMP Adenosine 3′:5′-cyclic monophosphate    -   DMEM Dulbecco's Modified Eagle's Medium    -   DMSO Dimethyl sulfoxide    -   EDTA Ethylenediaminetetraacetic acid    -   Emax maximal efficacy    -   FBS Fetal bovine serum    -   Gly Glycine    -   HEK-293 Human embryonic kidney-293    -   PBS Phosphate buffered saline    -   rpm rotations per minute    -   Tris Tris(hydroxymethyl)aminomethane

Membrane Preparation from Cells Expressing Human β₁ or β₂ AdrenergicReceptors

HEK-293 derived cell lines stably expressing cloned human β₁ or β₂adrenergic receptors, respectively, were grown to near confluency inDMEM with 10% dialyzed FBS in the presence of 500 μg/mL Geneticin. Thecell monolayer was lifted with Versene 1:5,000 (0.2 g/L EDTA in PBS)using a cell scraper. Cells were pelleted by centrifugation at 1,000rpm, and cell pellets were either stored frozen at −80° C. or membraneswere prepared immediately. For preparation, cell pellets wereresuspended in lysis buffer (10 mM Tris/HCL pH 7.4 @4° C., one tablet of“Complete Protease Inhibitor Cocktail Tablets with 2 mM EDTA” per 50 mLbuffer (Roche cat. #1697498, Roche Molecular Biochemicals, Indianapolis,Ind.)) and homogenized using a tight-fitting Dounce glass homogenizer(20 strokes) on ice. The homogenate was centrifuged at 20,000×g, thepellet was washed once with lysis buffer by resuspension andcentrifugation as above. The final pellet was resuspended in membranebuffer (75 mM Tris/HCl pH 7.4, 12.5 mM MgCl₂, 1 mM EDTA @25° C.).Protein concentration of the membrane suspension was determined by themethod of Bradford (Bradford M M., Analytical Biochemistry, 1976, 72,248-54). Membranes were stored frozen in aliquots at −80° C.

Test A Radioligand Binding Assay on Human β₁ and β₂ Adrenergic Receptors

Binding assays were performed in 96-well microtiter plates in a totalassay volume of 100 μL with 5 μg membrane protein for membranescontaining the human β₂ adrenergic receptor, or 2.5 μg membrane proteinfor membranes containing the human μ₁ adrenergic receptor in assaybuffer (75 mM Tris/HCl pH 7.4 @25° C., 12.5 mM MgCl₂, 1 mM EDTA, 0.2%BSA). Saturation binding studies for determination of K_(d) values ofthe radioligand were done using [³H]dihydroalprenolol (NET-720, 100Ci/mmol, PerkinElmer Life Sciences Inc., Boston, Mass.) at 10 differentconcentrations ranging from 0.01 nM-200 nM. Displacement assays fordetermination of pK_(i) values of compounds were done with[³H]dihydroalprenolol at 1 nM and 10 different concentrations ofcompound ranging from 40 pM-10 μM. Compounds were dissolved to aconcentration of 10 mM in dissolving buffer (25 mM Gly-HCl pH 3.0 with50% DMSO), then diluted to 1 mM in 50 mM Gly-HCl pH 3.0, and from thereserially diluted into assay buffer. Non-specific binding was determinedin the presence of 10 μM unlabeled alprenolol. Assays were incubated for90 minutes at room temperature, binding reactions were terminated byrapid filtration over GF/B glass fiber filter plates (Packard BioScienceCo., Meriden, Conn.) presoaked in 0.3% polyethyleneimine. Filter plateswere washed three times with filtration buffer (75 mM Tris/HCl pH 7.4@4° C., 12.5 mM MgCl₂, 1 mM EDTA) to remove unbound radioactivity.Plates were dried, 50 μL Microscint-20 liquid scintillation fluid(Packard BioScience Co., Meriden, Conn.) was added and plates werecounted in a Packard Topcount liquid scintillation counter (PackardBioScience Co., Meriden, Conn.). Binding data were analyzed by nonlinearregression analysis with the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.) using the 3-parameter model forone-site competition. The curve minimum was fixed to the value fornonspecific binding, as determined in the presence of 10 μM alprenolol.K_(i) values for compounds were calculated from observed IC₅₀ values andthe K_(d) value of the radioligand using the Cheng-Prusoff equation(Cheng Y, and Prusoff W H., Biochemical Pharmacology, 1973, 22, 23,3099-108). The receptor subtype selectivity was calculated as the ratioof K_(i)(β₁)/K_(i)(β₂). Compounds of the invention demonstrated greaterbinding at the β₂ adrenergic receptor than at the β₁ adrenergicreceptor, i.e. K_(i)(β₁)>K_(i)(β₂) with selectivity greater than about30.

Test B Whole-cell cAMP Flashplate Assays with Cell Lines HeterologouslyExpressing Human β₁ Adrenoceptor, β₂ Adrenoceptor, and β₃ Adrenoceptor,Respectively

A HEK-293 cell line stably expressing cloned human β₁ adrenergicreceptor (clone H34.1) was grown to about 70%-90% confluency in mediumconsisting of DMEM supplemented with 10% FBS and 500 μg/mL Geneticin. AHEK-293 cell line stably expressing cloned human β₂-adrenoceptor (cloneH24.14) was grown in the same medium to full confluency. A CHO-K1 cellline stably expressing cloned human β₃-adrenoceptor was grown to about70%-90% confluency in Ham's F-12 medium supplemented with 10% FBS andwith 800 μg/mL Geneticin added to every fifth passage. The day beforethe assay, cultures were switched to the same growth-media withoutantibiotics.

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), according to themanufacturers instructions.

On the day of the assay, cells were rinsed once with PBS, lifted withVersene 1:5,000 (0.2 g/L EDTA in PBS) and counted. Cells were pelletedby centrifugation at 1,000 rpm and resuspended in stimulation bufferprewarmed to 37° C. For cells expressing the β₁-adrenoceptor, 10 nM ICI118,551 were added to the stimulation buffer, and cells were incubatedfor 10 mM at 37° C. Cells were used at final concentrations of 30,000,40,000 and 70,000 cells/well for the β₁-adrenoceptor-, theβ₂-adrenoceptor- and the β₃-adrenoceptor expressing cells, respectively.Compounds were dissolved to a concentration of 10 mM in DMSO, thendiluted to 1 mM in 50 mM Gly-HCl pH 3.0, and from there serially dilutedinto assay buffer (75 mM Tris/HCl pH 7.4 @25° C., 12.5 mM MgCl₂, 1 mMEDTA, 0.2% BSA). Compounds were tested in the assay at 11 differentconcentrations, ranging from 10 μM to 9.5 pM. Reactions were incubatedfor 10 min at 37° C. and stopped by addition of 100 μl ice-colddetection buffer. Plates were sealed, incubated over night at 4° C. andcounted the next morning in a topcount scintillation counter (PackardBioScience Co., Meriden, Conn.). The amount of cAMP produced per mL ofreaction was calculated based on the counts observed for the samples andcAMP standards, as described in the manufacturer's user manual. Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using the3-parameter model for sigmoidal dose-response (Hill slope=1). Agonistpotencies were expressed as pEC₅₀ values.

Compounds of the invention demonstrated potent activity at the β₂adrenergic receptor in this assay, as evidenced by pEC₅₀ values greaterthan about 9. In addition, the compounds tested demonstrated selectivityin functional activity at the β₂ receptor as compared with functionalactivity at the β₁ and β₃ receptors. In particular, compounds of theinvention demonstrated EC₅₀(β₁)/EC₅₀(β₂) ratios of greater than about 10and EC₅₀(β₃)/EC₅₀(β₂) ratios of greater than about 50.

Test C Whole-Cell cAMP Flashplate Assay With a Lung Epithelial Cell LineEndogenously Expressing Human β₂ Adrenergic Receptor

For the determination of agonist potencies and efficacies (intrinsicactivities) in a cell line expressing endogenous levels of β₂ adrenergicreceptor, a human lung epithelial cell line (BEAS-2B) was used (ATCCCRL-9609, American Type Culture Collection, Manassas, Va.) (January B,et al., British Journal of Pharmacology, 1998, 123, 4, 701-11). Cellswere grown to 75-90% confluency in complete, serum-free medium (LHC-9MEDIUM containing Epinephrine and Retinoic Acid, cat #181-500, BiosourceInternational, Camarillo, Calif.). The day before the assay, medium wasswitched to LHC-8 (No epinephrine or retinoic acid, cat #141-500,Biosource International, Camarillo, Calif.).

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), according to themanufacturers instructions.

On the day of the assay, cells were rinsed with PBS, lifted by scrapingwith 5 mM EDTA in PBS, and counted. Cells were pelleted bycentrifugation at 1,000 rpm and resuspended in stimulation bufferprewarmed to 37° C. at a final concentration of 600,000 cells/mL. Cellswere used at a final concentration of 30,000 cells/well in the assay.Compounds were dissolved to a concentration of 10 mM in dissolvingbuffer (25 mM Gly-HCl pH 3.0 with 50% DMSO), then diluted to 1 mM in 50mM. Gly-HCl pH 3.0, and from there serially diluted into assay buffer(75 mM Tris/HCl pH 7.4 @25° C., 12.5 mM MgCl₂, 1 mM EDTA, 0.2% BSA).

Compounds were tested in the assay at 10 different concentrations,ranging from 10 μM to 40 pM. Maximal response was determined in thepresence of 10 μM Isoproterenol. Reactions were incubated for 10 mM at37° C. and stopped by addition of 100 μl ice-cold detection buffer.Plates were sealed, incubated over night at 4° C. and counted the nextmorning in a topcount scintillation counter (Packard BioScience Co.,Meriden, Conn.). The amount of cAMP produced per mL of reaction wascalculated based on the counts observed for samples and cAMP standards,as described in the manufacturer's user manual. Data were analyzed bynonlinear regression analysis with the GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.) using the 4-parameter modelfor sigmoidal dose-response with variable slope. Compounds of theinvention tested in this assay demonstrated pEC₅₀ values greater thanabout 8.

Compound efficacy (% Eff) was calculated from the ratio of the observedEmax (TOP of the fitted curve) and the maximal response obtained for 10μM isoproterenol and was expressed as % Eff relative to isoproterenol.The compounds tested demonstrated a % Eff greater than about 50.

Test D Assay of Bronchoprotection Against Acetylcholine-InducedBronchospasm in a Guinea Pig Model

Groups of 6 male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g were individuallyidentified by cage cards. Throughout the study animals were allowedaccess to food and water ad libitum.

Test compounds were administered via inhalation over 10 minutes in awhole-body exposure dosing chamber (R&S Molds, San Carlos, Calif.). Thedosing chambers were arranged so that an aerosol was simultaneouslydelivered to 6 individual chambers from a central manifold. Following a60 minute acclimation period and a 10 minute exposure to nebulized waterfor injection (WFI), guinea pigs were exposed to an aerosol of testcompound or vehicle (WFI). These aerosols were generated from aqueoussolutions using an LC Star Nebulizer Set (Model 22F51, PARI RespiratoryEquipment, Inc. Midlothian, Va.) driven by a mixture of gases (CO₂=5%,O₂=21% and N₂=74%) at a pressure of 22 psi. The gas flow through thenebulizer at this operating pressure was approximately 3 L/minute. Thegenerated aerosols were driven into the chambers by positive pressure.No dilution air was used during the delivery of aerosolized solutions.During the 10 minute nebulization, approximately 1.8 mL of solution wasnebulized. This was measured gravimetrically by comparing pre-andpost-nebulization weights of the filled nebulizer.

The bronchoprotective effects of compounds administered via inhalationwere evaluated using whole body plethysmography at 1.5, 24, 48 and 72hours post-dose. Forty-five minutes prior to the start of the pulmonaryevaluation, each guinea pig was anesthetized with an intramuscularinjection of ketamine (43.75 mg/kg), xylazine (3.50 mg/kg) andacepromazine (1.05 mg/kg). After the surgical site was shaved andcleaned with 70% alcohol, a 2-5 cm midline incision of the ventralaspect of the neck was made. Then, the jugular vein was isolated andcannulated with a saline-filled polyethylene catheter (PE-50, BectonDickinson, Sparks, Md.) to allow for intravenous infusions of a 0.1mg/mL solution of acetylcholine (Ach), (Sigma-Aldrich, St. Louis, Mo.)in saline. The trachea was then dissected free and cannulated with a 14Gteflon tube (#NE-014, Small Parts, Miami Lakes, Fla.). If required,anesthesia was maintained by additional intramuscular injections of theaforementioned anesthetic cocktail. The depth of anesthesia wasmonitored and adjusted if the animal responded to pinching of its paw orif the respiration rate was greater than 100 breaths/minute.

Once the cannulations were complete, the animal was placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula was inserted to measure pulmonary drivingpressure (pressure). The teflon tracheal tube was attached to theopening of the plethysmograph to allow the guinea pig to breathe roomair from outside the chamber. The chamber was then sealed. A heatinglamp was used to maintain body temperature and the guinea pig's lungswere inflated 3 times with 4 mL of air using a 10 mL calibration syringe(#5520 Series, Hans Rudolph, Kansas City, Mo.) to ensure that the lowerairways had not collapsed and that the animal did not suffer fromhyperventilation.

Once it was determined that baseline values were within the range0.3-0.9 mL/cm H₂O for compliance and within the range 0.1-0.199 cmH₂O/mL per second for resistance, the pulmonary evaluation wasinitiated. A Buxco pulmonary measurement computer program enabled thecollection and derivation of pulmonary values. Starting this programinitiated the experimental protocol and data collection. The changes involume over time that occurred within the plethysmograph with eachbreath were measured via a Buxco pressure transducer. By integratingthis signal over time, a measurement of flow was calculated for eachbreath. This signal, together with the pulmonary driving pressurechanges, which were collected using a Sensym pressure transducer(#TRD4100), was connected via a Buxco (MAX 2270) preamplifier to a datacollection interface (#'s SFT3400 and SFT3813). All other pulmonaryparameters were derived from these two inputs.

Baseline values were collected for 5 minutes, after which time theguinea pigs were challenged with Ach. Ach was infused intravenously for1 minute from a syringe pump (sp210iw, World Precision Instruments,Inc., Sarasota, Fla.) at the following doses and prescribed times fromthe start of the experiment: 1.9 μg/minute at 5 minutes, 3.8 μg/minuteat 10 minutes, 7.5 μg/minute at 15 minutes, 15.0 μg/minute at 20minutes, 30 μg/minute at 25 minutes and 60 μg/minute at 30 minutes. Ifresistance or compliance had not returned to baseline values at 3minutes following each Ach dose, the guinea pig's lungs were inflated 3times with 4 mL of air from a 10 mL calibration syringe. Recordedpulmonary parameters included respiration frequency (breaths/minute),compliance (mL/cm H₂O) and pulmonary resistance (cm H₂O/mL per second)(Giles et al., 1971). Once the pulmonary function measurements werecompleted at minute 35 of this protocol, the guinea pig was removed fromthe plethysmograph and euthanized by CO₂ asphyxiation.

The quantity PD₂, which is defined as the amount of Ach needed to causea doubling of the baseline pulmonary resistance, was calculated usingthe pulmonary resistance values derived from the flow and the pressureover a range of Ach challenges using the following equation. This wasderived from the equation used to calculate PC₂₀ values in the clinic(Am. Thoracic Soc, 2000).

${PD}_{2} = {{antilog}\left\lbrack {{\log\; C_{1}} + \frac{\left( {{\log\; C_{2}} - {\log\; C_{1}}} \right)\left( {{2R_{0}} - R_{1}} \right)}{R_{2} - R_{1}}} \right\rbrack}$

where:

C₁=Second to last Ach concentration (concentration preceding C₂)

C₂=Final concentration of Ach (concentration resulting in a 2-foldincrease in pulmonary resistance (R_(L)))

R₀=Baseline R_(L) value

R₁=R_(L) value after C₁

R₂=R_(L) value after C₂

Statistical analysis of the data was performed using a One-Way Analysisof Variance followed by post-hoc analysis using a Bonferroni/Dunn test.A P-value<0.05 was considered significant.

Dose-response curves were fitted with a four parameter logistic equationusing GraphPad Prism, version 3.00 for Windows (GraphPad Software, SanDiego, Calif.)Y=Min+(Max−Min)/(1+10^((log ED₅₀−X)* Hillslope)),

where X is the logarithm of dose, Y is the response (PD₂), and Y startsat Min and approaches asymptotically to Max with a sigmoidal shape.

Representative compounds of the invention were found to have significantbronchoprotective activity at time points beyond 24 hours post-dose.

The following synthetic examples are offered to illustrate theinvention, and are not to be construed in any way as limiting the scopeof the invention.

EXAMPLES

General: Unless noted otherwise, reagents, starting material andsolvents were purchased from commercial suppliers, for exampleSigma-Aldrich (St. Louis, Mo.), J. T. Baker (Phillipsburg, N.J.), andHoneywell Burdick and Jackson (Muskegon, Mich.), and used withoutfurther purification; reactions were run under nitrogen atmosphere;reaction mixtures were monitored by thin layer chromatography (silicaTLC), analytical high performance liquid chromatography (anal. HPLC), ormass spectrometry; reaction mixtures were commonly purified by flashcolumn chromatography on silica gel, or by preparative HPLC using thegeneral protocol described below; NMR samples were dissolved indeuterated solvent (CD₃OD, CDCl₃, or DMSO-d6), and spectra were acquiredwith a Varian Gemini 2000 instrument (300 MHz) under standardparameters; and mass spectrometric identification was performed by anelectrospray ionization method (ESMS) with a Perkin Elmer instrument (PESCIEX API 150 EX).

Example 1 Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onea. Preparation of 4-(2-amino-2-methyl-propoxy)-phenylamine hydrochloride

A vigorously stirred slurry of sodium hydride (60% dispersion in mineraloil, 11.32 g, 0.28 mol) in dimethylsulfoxide (400 mL) was heated at 45°C. for 1 h. To this slurry was then added neat2-amino-2-methyl-1-propanol (25.3 g, 1 equiv). The reaction mixture waswarmed to 75° C. for 1 h then cooled to 20° C. in an ice bath.1-Fluoro-4-nitrobenzene (40 g, 1 equiv) was added slowly, maintainingthe temperature below 30° C., and the resulting dark red solution wasstirred at room temperature for a further 1 h. The reaction was quenchedwith water (1000 mL), extracted with dichloromethane (500 mL), and theorganic layer washed (1:1 saturated aqueous sodium chloride:water, 1000mL). The product was precipitated by addition of 3M hydrochloric acid(400 mL) to the organic layer. The resulting orange solid was thenfiltered and washed with dichloromethane until the filtrate wascolorless.

The solid material was immediately transferred to a hydrogenation flask.Palladium (10% w/w on carbon, 50% w/w water) was added, followed bymethanol (500 mL). The slurry was shaken vigorously under 3 atmospheresof hydrogen gas for 16 h. The catalyst was then filtered, the solventremoved under reduced pressure, and the resulting solid dried byazeotroping with toluene (3×150 mL) to afford the title intermediate asa white solid (40 g, 0.18 mol, 65%.

b. Preparation of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-8-benzyloxy-1H-quinolin-2-one

A mixture of the product of step a (23.2 g, 1.1 equiv),8-benzyloxy-5-{(R)-2-[2-(4-bromo-phenyl)-ethylamino-1-(tert-butyl-dimethyl-silanyloxy)-ethyl}-1H-quinolin-2-onehydrochloride (66.0 g, 0.1 mol), and sodium tert-butoxide (54.0 g, 5.5equiv) in toluene (600 mL) was stirred at 90° C. until a homogenoussolution was obtained. Palladium tris(dibenzylideneacetone) (1.4 g,0.015 equiv) was added, followed byrac-2,2′-bis(diphenylphosphino)-1,1′-binapthyl (2.87 g, 0.045 equiv).The reaction mixture was stirred at 90° C. for 3 h, then allowed tocool. The solution was washed with water (100 mL), 1:1 saturated aqueoussodium chloride:water (100 mL), then dried over sodium sulfate. Thesolvent was removed under reduced pressure to afford the titleintermediate as a dark brown solid (40 g crude), which was used withoutfurther purification.

c. Preparation of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-benzyloxy-1H-quinolin-2-one

The product of the previous step was treated with triethylaminetrihydrofluoride (36 g) in 2-propanol (500 mL)/ethanol (100 mL) at roomtemperature for 16 h. The mixture was concentrated under reducedpressure to one third of its original volume. 1M aqueous sodiumhydroxide (500 mL) was added, followed by acetonitrile (500 mL) andisopropyl acetate (500 mL). The aqueous layer was removed and theorganic phase washed with 1:1 saturated aqueous sodium chloride:water(400 mL) then saturated aqueous sodium chloride (400 mL). The organicswere dried over sodium sulfate and the solvent removed in vacuo toafford the title intermediate (50 g crude) as a brown solid, which wasused without further purification.

d. Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one

Palladium hydroxide (10 g, 20% w/w on carbon, 50% w/w water) was addedto the product from the previous reaction, followed by ethanol (500 mL).The slurry was stirred vigorously under an atmosphere of hydrogen gasfor 8 h. The catalyst was filtered and the filtrate concentrated underreduced pressure to afford the title compound (40 g), which was purifiedby reverse phase HPLC and isolated as its trifluoroacetate salt bylyophilization. ¹H NMR (300 MHz, DMSO-d₆): 10.4 (s, 1H), 9.3 (br s, 1H),8.7 (br s, 1H), 8.15 (m, 2H), 7.8 (br s, 1H), 7.03 (d, 1H, J=8.2),6.76-7.01 (m, 10H), 6.42 (d, 1H, J=9.6), 6.1 (br s, 1H), 5.33 (d, 1H,J=9.1), 3.8 (s, 2H), 2.7-3.1 (m, 6H), 1.21 (s, 6H); m/z: [M+H⁺] calcdfor C₂₉H₃₄N₄O₄, 503.3; found 503.5.

Examples 2-8 Synthesis of Compounds 2-8

Using procedures similar to those described in Example 1, exceptreplacing the 2-amino-2-methyl-1-propanol with the appropriate alcoholin step a, trifluoroacetate salts of compounds 2-8 were prepared.

-   Compound 2: 5-[(R)-2-(2-{4-[4-(2-amino-ethoxy)    -phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one:    m/z: [M+H⁺] calcd for C₂₇H₃₁N₄O₄, 475.2; found 475.3.-   Compound 3: 5-[(R)-2-(2-{4-[4-(3-amino-propoxy)    -phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one:    m/z: [M+H⁺] calcd for C₂₈H₃₃N₄O₄, 489.2; found 489.5.-   Compound 4: 5-[(R)-2-(2-{4-[4-(4-amino-butoxy)    -phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one:    m/z: [M+H⁺] calcd for C₂₉H₃₅N₄O₄, 503.3; found 503.5.-   Compound 5:    5-{(R)-2-[2-(4-{4-[2-(2-amino-ethoxy)-ethoxy]-phenylamino}-phenyl)-ethylamino]-1-hydroxy-ethyl}-8-hydroxy-1H-quinolin-2-one:    m/z: [M+H⁺] calcd for C₂₉H₃₅N₄O₅, 519.3; found 519.5.-   Compound 6:    8-hydroxy-5-[(R)-1-hydroxy-2-(2-{4-[4-(2-morpholin-4-yl-ethoxy)-phenylamino]-phenyl}-ethylamino)-ethyl]-1H-quinolin-2-one:    m/z: [M+H⁺] calcd for C₃₁H₃₇N₄O₅, 545.3; found 545.6.-   Compound 7:    8-hydroxy-5-[(R)-1-hydroxy-2-(2-{4-[4-(2-piperazin-1-yl-ethoxy)-phenylamino]-phenyl}-ethylamino)-ethyl]-1H-quinolin-2-one:    m/z: [M+H⁺] calcd for C₃₁H₃₈N₅O₄, 544.7; found 544.7.-   Compound 8:    8-hydroxy-5-[(R)-1-hydroxy-2-(2-{4-[4-pyridin-2-ylmethoxy)-phenylamino]-phenyl}-ethylamino)-ethyl]-1H-quinolin-2-one:    m/z: [M+H⁺] calcd for C₃₁H₃₁N₄O₄, 523.1; found 523.2.

Example 9 Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-3-trifluoromethyl-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one

Using procedures similar to those described for Example 1, exceptreplacing the 1-fluoro-4-nitrobenzene with1-fluoro-4-nitro-2-trifluoromethylbenzene in step a, the title compoundwas prepared. m/z: [M+H⁺] calcd for C₃₀H₃₄F₃N₄O₄, 571.3; found 571.3.

Example 10 Synthesis of8-hydroxy-5-{(R)-1-hydroxy-2-[2-(4-{4-[2-(4-methanesulfonyl-piperazin-1-yl)-ethoxy]-phenylamino}-phenyl)-ethylamino]-ethyl}-1H-quinolin-2-onea. Preparation of 8-benzyloxy-5-[(R)-1-(tert-butyl-dimethyl-silanyloxy)-2-(2-{4-[4-(2-piperazin-1-yl-ethoxy)-phenylamino]-phenyl}-ethylamino)-ethyl]-1H-quinolin-2-one

Using procedures similar to those described for Example 1, steps a andb, except replacing the 2-amino-2-methyl-1-propanol withN-(2-hydroxyethyl)piperazine in step a, the title intermediate wasprepared.

b. Preparation of 8-benzyloxy-5-{(R)-1-(tert-butyl-dimethyl-silanyloxy)-2-[2-(4-{4-[2-(4-methanesulfonyl-piperazin-1-yl)-ethoxy]-phenylamino}-phenyl)-ethylamino]-ethyl}-1H-quinolin-2-one

The product of step a (100 mg) was treated with methanesulfonyl chloride(15.3 mg, 1 equiv) in a mixture of diisopropylethylamine (0.14 mL) andtetrahydrofuran (2 mL) at room temperature for 1 h. The mixture was thenevaporated to yield the title intermediate.

c. Synthesis of8-hydroxy-5-{(R)-1-hydroxy-2-[2-(4-{4-[2-(4-methanesulfonyl-piperazin-1-yl)-ethoxy]-phenylamino}-phenyl)-ethylamino]-ethyl}-1H-quinolin-2-one

Using procedures similar to those described for Example 1, steps c andd, the intermediate of step b was transformed to the title compound.m/z: [M+H⁺]calcd for C₃₂H₄₀N₅O₆S, 622.3; found 622.5.

Example 11 Synthesis of 5-1-[(R)-2-(2-{4-[4-(2-amino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onea. Preparation of 5-[(R)-2-(2-{4-[4-(2-amino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-8-benzyloxy-1H-quinolin-2-one

Tris(dibenzylideneacetone)dipalladium(0) (0.028 g, 0.031 mmol), followedby rac-2,2′-bis(diphenylphosphino)-1,1′-binapthyl (0.057 g, 0.092 mmol)was added to a solution of 2-(4-aminophenyl)ethylamine (0.100 g, 0.740mmol),8-benzyloxy-5-{(R)-2-[2-(4-bromo-phenyl)-ethylamino-1-(tert-butyl-dimethyl-silanyloxy)-ethyl}-1H-quinolin-2-onehydrochloride (0.393 g, 0.612 mmol), and sodium tert-butoxide (0.265 g,2.75 mmol) in toluene (20 mL) at room temperature. The resulting mixturewas heated at 90° C. for 2 h, then allowed to cool. The solution waswashed with water (100 mL), 1:1 saturated aqueous sodium chloride:water(100 mL), then dried over magnesium sulfate. The solvent was removed atreduced pressure to afford the title intermediate as a dark brown solid(0.473 g), which was used without further purification.

b. Preparation of 5-[(R)-2-(2-{4-[4-(2-amino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-benzyloxy-1H-quinolin-2-one

The product of the previous step (0.473 g, 0.714 mmol) was treated withtriethylamine trihydrofluoride (0.173 g, 1.07 mmol) in tetrahydrofuran(20 mL) at room temperature for 16 h. The mixture was diluted withdichloromethane (100 mL) and water (100 mL). The resulting mixture wasstirred vigorously and made basic (pH>10) by the addition of 1N aqueoussodium hydroxide. The organic phase was washed with water (200 mL) andthen washed with saturated aqueous sodium chloride (200 mL). Theorganics were dried over magnesium sulfate and the solvent removed atreduced pressure to afford the title intermediate (0.500 g) as a brownsolid, which was used without further purification.

c. Synthesis of 5-[(R)-2-(2-{4-[4-(2-amino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one

Palladium (0.100 g, 10% wt. on activated carbon) was added to a solutionof the product of the previous step in 1:1 methanol:dichloromethane (20mL). The slurry was stirred vigorously under 1 atmosphere of hydrogenfor 16 h. The reaction mixture was filtered through celite andconcentrated at reduced pressure to afford the title compound, which waspurified by reverse phase HPLC and isolated as its trifluoroacetate saltby lyophilization. ¹H NMR (300 MHz, DMSO-d₆): δ 10.4 (d, 2H), 8.6 (br s,2H), 8.0 (d, 2H), 7.7 (br s, 3H), 7.0 (d, 1H), 6.96 (d, 4H), 6.84-6.88(m, 4H), 6.45 (d, 1H), 6.08 (s, 1H), 5.19 (d, 1H), 3.0-3.2 (m, 2H), 2.8(br s, 2H), 2.72-2.75 (m, 2H), 2.62 (m, 2H); m/z: [M+H⁺] calcd forC₂₇H₃₀N₄O₃, 458.56; found 459.4.

Example 12 Synthesis of 5-[(R)-2-(2-{4-[4-(2-dimethylamino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onea. Preparation of dimethyl-[2-(4-nitro-phenyl)-ethyl]-amine

Dimethylamine hydrochloride (0.390 g, 4.78 mmol) was added to a solutioncontaining 4-nitrophenethyl bromide (1.0 g, 4.35 mmol) and N,N-diisopropylethyamine (1.69 g, 13.05 mmol) in anhydrousdimethylformamide (20 mL). The reaction mixture was heated to 60° C. for5 hours, then allowed to cool. The solution was diluted with 1:1water:dichloromethane (200 mL), then added to a separatory funnel. Theorganics were collected and the product was extracted with 1N aqueoushydrogen chloride. The organics were removed and the aqueous layer wasmade basic with 1N aqueous sodium hydroxide. The product was extractedwith dichloromethane (100 mL) and washed with saturated aqueous sodiumchloride (200 mL). The organics were dried over magnesium sulfate andthe solvent removed at reduced pressure to afford the title intermediate(0.426 g crude) as a clear oil, which was used without furtherpurification.

b. Preparation of 4-(2-dimethylamino-ethyl)-phenylamine

Palladium (0.043 g, 10% wt. on activated carbon) was added to a solutionof the product of the previous step (0.430 g, 2.2 mmol) in methanol (20mL). The slurry was stirred vigorously under a hydrogen atmosphere for 6hours. The reaction mixture was filtered and the filtrate concentratedat reduced pressure to afford the title intermediate (0.307 g crude)which was used without further purification.

c. Synthesis of5-[(R)-2-(2-{4-[4-(2-dimethylamino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one

Using procedures similar to those described for Example 11, exceptreplacing the 2-(4-aminophenyl)ethylamine of Example 11, step a, withthe product of the previous step, the trifluoroacetate salt of the titlecompound was prepared. ¹H NMR (300 MHz, DMSO-d₆): δ 10.4 (s, 2H), 9.8(br s, 1H), 8.9 (br s, 1H), 8.7 (br s, 1H), 8.0 (d, 2H), 6.8-7.2 (m,10H), 6.4 (d, 1H), 6.2 (br s, 1H), 5.2 (d, 1H), 2.8-3.1 (m, 6H),2.3-2.7(m, 8H); m/z: [M+H⁺] calcd for C₂₉H₃₄N₄O₃, 486.61; found 487.5.

Example 13 Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onea. Preparation of 1,1-dimethyl-2-(4-nitro-phenyl)-ethylamine

α,α-Dimethylphenethylamine hydrochloride (20 g, 108 mmol) was dissolvedin concentrated sulfuric acid (40 mL). The solution was cooled to −10°C. Nitric acid (31 mL, 90%) was added dropwise over a 30 min periodwhile maintaining a reaction tempurature below −5° C. The solution wasstirred an addition 45 min then poured over ice and allowed to warm toroom temperature over night. The product was collected via filtration(13.7 g). Aqueous sodium hydroxide(1 N) was added to the filtrate andthe title compound was isolated as a clear oil.

b. Preparation of 4-(2-amino-2-methyl-propyl)-phenylamine

Palladium (0.136 g, 10% wt. on activated carbon) was added to a solutionof the product of step a (1.36 g, 7.0 mmol) in methanol (20 mL). Theslurry was stirred vigorously under 1 atmosphere of hydrogen for 6 h.The reaction mixture was filtered and the filtrate concentrated underreduced pressure to afford the title intermediate (1.04 g crude) whichwas used without further purification.

c. Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one

Using procedures similar to those described for Example 11, exceptreplacing the 2-(4-aminophenyl)ethylamine of Example 11, step a, withthe product of the previous step, the trifluoroacetate salt of the titlecompound was prepared. ¹H NMR (300 MHz, DMSO-d₆): δ 10.4 (d, 2H), 8.8(br s, 1H), 8.65 (br s, 1H), 8.0 (d, 2H), 7.7 (s, 3H), 6.8-7.0 (m, 8H),6.4 (d, 1H), 6.1 (br s, 1H), 5.2 (d, 1H), 3 (br s, 4H), 2.7(m, 2H), 2.6(s, 2H), 1.0 (s, 6H); m/z: [M+H⁺] calcd for C₂₉H₃₄N₄O₃, 486.61; found487.5.

Example 14 Synthesis ofN-{5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-2-hydroxy-phenyl}-formamidea. Preparation of[4-(2-amino-2-methyl-propoxy-phenyl]-[4-(2-aminoethyl)-phenyl]-amine

A mixture of 4-(2-amino-2-methyl-propoxy)-phenylamine (1.0 g, 5.5 mmol)(the freebase of the intermediate of Example 1, part a),4-bromophenethylamine (1.1 g, 1 equiv), and sodium tert-butoxide (1.9 g,3.5 equiv) in toluene (30 mL) was stirred at 90° C. until a homogenoussolution was obtained. Tris(dibenzylideneacetone)dipalladium(0) (252 mg,0.05 equiv) was added, followed byrac-2,2′-bis(diphenylphosphino)-1,1′-binapthyl (516 mg, 0.015 equiv).The reaction mixture was stirred at 90° C. for 16 h, then allowed tocool. The solution was diluted with ethyl acetate (100 mL), washed (1:1saturated aqueous sodium chloride:water (100 mL), then extracted into 6M hydrochloric acid (100 mL). The aqueous layer was washed with ethylacetate (2×100 mL), then diluted with isopropyl acetate (100 mL). Themixture was cooled to 0° C. and neutralized with sodium hydroxide (13 gin 20 mL of water). The aqueous layer was removed and the organic layerwashed (1:1 saturated aqueous sodium chloride:water, 100 mL), dried oversodium sulfate, and evaporated to afford the title intermediate (1.3 gcrude).

b. Preparation ofN-{5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-2-benzyloxy-phenyl}-formamide

A mixture of N-[5-((R)-2-bromo-1-(tert-butyl-dimethyl-silanyloxy)-ethyl)-2-benzyloxy-phenyl]-formamide (2.0 g, 4.35 mmol), the product of theprevious step (1.3 g, 1 equiv), potassium carbonate (2.4 g, 4 equiv) andsodium iodide (718 mg, 1.1 equiv) in dimethylsulfoxide (8 mL) was heatedat 140° C. for 20 min. The mixture was allowed to cool, diluted withwater (20 mL) and extracted with ethyl acetate (2×20 mL). The organicswere washed with saturated aqueous sodium chloride (20 mL), dried oversodium sulfate, and evaporated. The residue was purified by reversephase HPLC to afford the title intermediate (500 mg, 0.73 mmol, 17%yield).

c. Synthesis of N-{5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-2-hydroxy-phenyl}-formamide

Using procedures similar to those described for Example 1, steps c andd, the intermediate of step b was transformed to the title compound.m/z: [M+H⁺] calcd for C₂₇H₃₅N₄O₄, 479.3; found 479.3.

Example 15 Synthesis of5-[(R)-2-(2-{4-[3-(2-amino-ethyl)-4-methoxy-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onea. Preparation of5-{(R)-2[2-(4-amino-phenyl)-ethylamino]-1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-8-benzyloxy-1H-quinolin-2-one

A mixture of5-(2-bromo-1-(tert-butyl-dimethyl-silanyloxy)-ethyl)-8-benzyloxy-1H-quinolin-2-one(5.0 g, 1.0 equiv) and 2-(4-aminophenyl)ethylamine (2.8 g, 2.0 equiv) indimethylsulfoxide (10 mL) was heated at 100° C. for 12 h. The reactionmixture was cooled to 20° C., and the resulting red oil was diluted withethyl acetate (200 mL) and extracted with two portions water (200 mL).The organic layer was washed with two portions of (1:1 10% aqueousacetic acid and saturated aqueous sodium chloride (200 mL). The organiclayer was basified by carefully extracting with two portions ofsaturated sodium bicarbonate (200 mL), followed by saturated sodiumchloride (200 mL). The resulting organic solution was treated withanhydrous sodium sulfate, and the solvent was removed under reducedpressure to afford the title intermediate (5.1 g) which was used withoutfurther purification.

b. Preparation of 5-[(R)-2-(2-{4-[3-(2-amino-ethyl)-4-methoxy-phenylamino]-phenyl}-ethylamino)-1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-8-benzyloxy-1H-quinolin-2-one

A mixture of the product of the previous step (0.500 g, 1.0 equiv),5-bromo -2-methoxyphenethylamine hydrobromide (0.343 g, 1.2 equiv), andsodium tert-butoxide (0.397 g, 4.5 equiv) in toluene (10 mL) was stirredat 90° C. until a homogenous solution was obtained. Palladiumtris(dibenzylideneacetone) (0.0042 g, 0.05 equiv) was added, followed byrac-2,2′-bis(diphenylphosphino)-1,1′-binapthyl (0.0086 g, 0.15 equiv).The reaction mixture was stirred at 90° C. for 5 h, then allowed to coolto 20° C. The solution was diluted with dichloromethane (100 mL) andwashed with water (2×100 mL). The organic solution was dried over sodiumsulfate. The reaction mixture was filtered and the solvent was removedunder reduced pressure to give the title intermediate (1.0 g) as a darkbrown solid, which was used without further purification.

c. Preparation of5-[(R)-2-(2-{4-[3-amino-ethyl)-4-methoxy-phenylamino]-phenyl}-ethylamino)-1-hydroxy)-ethyl]-8-benzyloxy-1H-quinolin-2-one

The crude solid (1.0 g) from the previous step was treated withtriethylamine trihydrofluoride (0.70 mL) in tetrahydrofuran (10 mL) atroom temperature for 5 h. The mixture was diluted with dichloromethane(100 mL) and extracted with 0.1 M NaOH (2×100 mL), followed by saturatedaqueous sodium chloride (100 mL). The organic layer was treated withanhydrous sodium sulfate and the solvent was dried under reducedpressure. The resulting solid was purified by reverse phase HPLC andisolated as its trifluoroacetate salt by lyophilization to give thetitle intermediate (200 mg).

d. Synthesis of 5-[(R)-2-(2-{4-[3-(2-amino-ethyl)-4-methoxy-phenylamino]-phenyl}-ethylamino)-1-hydroxy)-ethyl]-8-hydroxy-1H-quinolin-2-one

Palladium (10 mg, 10% w/w on carbon) followed by methanol (5 mL) wasadded to the intermediate of the previous step (50 mg) and the slurrywas stirred vigorously under a hydrogen atmosphere for 5 h. The reactionmixture was filtered and the filtrate was concentrated under reducedpressure to afford the title compound (42.0 mg), which was purified byreverse phase HPLC and isolated as its trifluoroacetate salt bylyophilization. m/z: [M+H⁺] calcd for C₂₈H₃₂N₄O₄, 489.3; found 489.5

Example 16 Synthesis of N-{5-[(R)-2-(2-{4-[3-(2-amino-ethyl)-4-methoxy-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-2-hydroxy-phenyl}-formamide

Using procedures similar to those described for Example 15, exceptreplacing the5-(2-bromo-1-(tert-butyl-dimethyl-silanyloxy)-ethyl)-8-benzyloxy-1H-quinolin-2-onewithN-[5-((R)-2-bromo-1-(tert-butyl-dimethyl-silanyloxy)-ethyl)-2-benzyloxy-phenyl]-formamide,the title compound was prepared. m/z: [M+H⁺] calcd for C₂₆H₃₂N₄O₄,465.3; found 465.3

Example 17 Salts of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onea. Preparation of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onefree base

Aqueous ammonium bicarbonate (10%) solution (50 mL) was added in oneportion to a solution of the product of Example 1,5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onetrifluoroacetate, (1.80 g, 2.1 mmol) in ethanol (6 mL). The solution wasstirred at room temperature for 1 h. The resultant solid was filteredand dried under reduced pressure to afford the title compound (0.84 g,1.67 mmol, 80%) as a yellow solid.

b. Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onesulfate

The free base produced as in the previous step (5.85 g, 11.6 mmol) wasdissolved in acetonitrile:water (200 mL:60 mL) at 90° C. and filtered toremove remaining solid particles. The filtrate was re-heated to 90° C.and a solution of concentrated sulfuric acid (2 mL) inacetonitrile:water (18 mL:2 mL) was added. The solution was allowed tocool to room temperature over 2 h, then cooled in an ice/water bath to10° C. The solid was filtered and dried under reduced pressure to affordthe crude title salt (5.70 g, 82%). The material (5.70 g) wasre-dissolved in acetonitrile:water (120 mL:410 mL) at 90 ° C., and againallowed to cool to room temperature over 2 h. The slurry was cooled inan ice/water bath to 10° C., the solid filtered and dried under reducedpressure to afford a hydrate of the title salt (4.40 g, 63% overall) asan off-white powder. The x-ray powder diffraction (XRPD) pattern of theproduct is shown in FIG. 1. The differential scanning calorimetry traceof the product is shown in FIG. 2.

c. Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onenapadisylate

To a solution of the free base produced as in part a (23 mg, 0.046 mmol)in isopropanol:water (1.8 mL:0.2 mL) at 60° C., was added1,5-napthalene-disulfonic acid tetrahydrate (33 mg, 2 equiv), affordinga white precipitate. The temperature was raised to 70° C. and another 1mL of water was added to afford a clear solution. After cooling to roomtemperature, the slurry was filtered and dried to afford the title salt(25 mg, 63%) as a white powder. The XRPD pattern of the product is shownin FIG. 3. The differential scanning calorimetry trace of the product isshown in FIG. 4.

d. Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-oneoxalate

To a solution of the free base produced as in part a (100 mg, 0.2 mmol)in isopropanol:water (3.6 mL:0.4 mL) at 60° C. was added oxalic acid (50mg, 2 equiv), affording a white precipitate. The temperature was raisedto 70° C. and another 4 mL of water was added to afford a clearsolution. After cooling to room temperature, the slurry was filtered anddried to afford the title salt (82 mg, 65%) as an off-white powder. TheXRPD pattern of the product is shown in FIG. 5. The differentialscanning calorimetry trace of the product is shown in FIG. 6.

e. Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methyl-cinnamate

5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one(0.21 g) was dissolved in a mixture of tetrahydrofuran (1.2 mL) andwater (1.2 mL). 4-methyl-cinnamic acid (0.07 g, predominantly trans) wasadded to the stirred solution at room temperature and it dissolved.After approximately 10 min, crystallization occurred. The slurry wasstirred overnight and filtered. The cake was washed with aqueoustetrahydrofuran (1:1 THF:water, 2×0.4 mL, 1×0.2 mL) to afford the titlecompound which was dried overnight under vacuum at 45° C. (yield: 0.207g). ¹H NMR (400 MHz, DMSO-d₆; DMSO-d₅ as reference at δ (ppm) 2.5). δ(ppm): 1.22 (6H) s; 2.30 (3H) s; 2.64 (2H) t J=6.6 Hz; 2.72-2.86 (4H) m;3.74 (2H) s; 5.09 (1H) m; 6.43 (1H) d J=15.9 Hz; 6.49 (1H) d J=10.0 Hz;6.86 (4H) m; 6.93-7.03 (5H) m; 7.06 (1H) d J=7.8 Hz; 7.17 (2H) d J=7.8Hz; 7.34 (1H) d J=15.9 Hz; 7.45 (2H) d J=7.8 Hz; 7.75 (1H) s; 8.19 (1H)d J=10.0 Hz. The crystalline product was characterized by XRPD and DSC.

f. Synthesis of5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one4-methyl-cinnamate

5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one(17.77 g) was dissolved in a mixture of tetrahydrofuran (89 mL) andwater (89 mL). 4-methyl-cinnamic acid (6.02 g, predominantly trans) wasweighed out and about one-quarter was added to the free base solutionfollowed by seed crystals, obtained by the procedure of part e. Themixture was stirred and the remaining acid was added in portions overthe following 1.5 h. The slurry was stirred for a further 4 h and thenthe slurry was filtered. The cake was washed with aqueoustetrahydrofuran (1:1 THF:water, 36 mL) and then with tetrahydrofuran(2×18 mL) to afford the title compound which was dried overnight undervacuum at 40-50° C. (yield: 16.916 g). ¹H NMR (400 MHz, CD₃OD; TMS asreference at δ (ppm) 0) δ (ppm): 1.39 (6H) s; 2.32 (3H) s; 2.79 (2H) tJ=7.2 Hz; 2.92-3.03 (4H) m; 3.87 (2H) s; 5.25 (1H) d of d J=3.9 and 8.8Hz; 6.44 (1H) d J=15.9 Hz; 6.63 (1H) d J=9.8 Hz; 6.86-6.93 (4H) m; 6.96(1H) d J=8.3 Hz; 6.99-7.05 (4H) m; 7.15 (2H) d J=7.8 Hz; 7.19 (1H) dJ=8.3 Hz; 7.36 (1H) d J=15.9 Hz; 7.38 (2H) d J=7.6 Hz; 8.34 (1H) d J=9.8Hz. The crystalline product was characterized by XRPD and DSC.

g. Analytical Methods

X-ray powder diffraction patterns of FIGS. 1, 3, and 5 were obtainedwith a Rigaku diffractometer using Cu Kα (30.0 kV, 15.0 mA) radiation.The analysis was performed with the goniometer running incontinuous-scan mode of 3° per min with a step size of 0.03° over arange of 2 to 45°. Samples were prepared on quartz specimen holders as athin layer of powdered material. The instrument was calibrated with asilicon metal standard.

Differential scanning calorimetry traces of FIGS. 2, 4, and 6 wereobtained with a TA instruments model DSCQ10. Samples were placed insealed aluminum pans for analysis with an empty pan serving as thereference. Samples were equilibrated at 30° C. and heated at 5° C. perminute to a temperature of 300° C. The instrument was calibrated with anindium standard.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

1. A method of treating asthma or chronic obstructive pulmonary diseasein a mammal, the method comprising administering to the mammal apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of formula (II):

wherein: R¹ and R² taken together are —NHC(═O)CH═CH— or —CH═CHC(═O)NH—;one of R⁵ and R⁶ is —[O—C₁₋₆alkylenyl]_(n)-NR¹⁰R¹¹ orC₁₋₆alkylenyl-NR¹²R¹³, and the other of R⁵ and R⁶ is selected fromhydrogen, hydroxy, C₁₋₄alkoxy, and C₁₋₄alkyl, wherein C₁₋₄alkyl isoptionally substituted with halo, wherein each of R¹⁰, R¹¹, R¹², and R¹³is independently hydrogen or C₁₋₄alkyl; or R¹⁰ and R¹¹, together withthe nitrogen atom to which they are attached, or R¹⁰, together with thenitrogen atom to which it is attached and a carbon atom of the adjacentC₁₋₆alkylenyl, or R¹² and R¹³, together with the nitrogen atom to whichthey are attached, or R¹², together with the nitrogen atom to which itis attached and a carbon atom of the adjacent C₁₋₆alkylenyl, form aheterocyclic or heteroaryl ring having from 5 to 7 ring atoms andoptionally containing an additional heteroatom selected from oxygen,nitrogen, and sulfur, wherein nitrogen is optionally substituted with—S(O)₂—C₁₋₄alkyl; and n is 1 or 2; or a pharmaceutically-acceptable saltor stereoisomer thereof.
 2. The method of claim 1 wherein: R⁵ isselected from —O—C₁₋₆alkylenyl-NR¹⁰R¹¹ and C₁₋₆alkylenyl-NR¹²R¹³ and R⁶is hydrogen; or R⁵ is C₁₋₄alkoxy and R⁶ is —C₁₋₆alkylenyl-NR¹²R¹³,wherein each of R¹⁰, R¹¹, R¹² and R¹³ is independently hydrogen orC₁₋₄alkyl, or R¹⁰ and R¹¹, together with the nitrogen atom to which theyare attached, form a piperazinyl ring.
 3. The method of claim 2 whereinR⁵ is —O—C₁₋₆alkylenyl-NR¹⁰R¹¹ and R⁶ is hydrogen.
 4. The method ofclaim 1 wherein the stereochemistry at the alkylene carbon bearing thehydroxyl group is (R).
 5. The method of claim 1 wherein the compound offormula (II) is selected from: 5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;8-hydroxy-5-[(R)-1-hydroxy-2-(2-{4-[4-(2-piperazin-1-yl-ethoxy)-phenylamino]-phenyl}-ethylamino)-ethyl]-1H-quinolin-2-one;5-[(R)-2-(2-{4-[4-(2-amino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;5-[(R)-2-(2-{4-[4-(2-dimethylamino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;and 5-[(R)-2-(2-{4-[3-(2amino-ethyl)-4-methoxy-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;and pharmaceutically-acceptable salts thereof.
 6. The method of claim 1wherein the compound of formula (II) is5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one,or a pharmaceutically-acceptable salt thereof.
 7. The method of claim 6wherein the compound of formula (II) is5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onesulfate.
 8. The method of claim 6 wherein the compound of formula (II)is5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-onenapadisylate.
 9. The method of claim 6 wherein the compound of formula(II) is5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-oneoxalate.
 10. The method of claim 1 wherein the method comprisingadministering the compound of formula (II) by inhalation.
 11. The methodof claim 1 further comprising administering one or more othertherapeutic agents wherein the other therapeutic agent is acorticosteroid or an anticholinergic agent.
 12. A method of providingbronchoprotection in a mammal, the method comprising administering tothe mammal a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a compound of formula (II):

wherein: R¹ and R² taken together are —NHC(═O)CH═CH— or —CH═CHC(═O)NH—;one of R⁵ and R⁶ is —[O—C₁₋₆alkylenyl]_(n)-NR¹⁰R¹¹ orC₁₋₆alkylenyl-NR¹²R¹³, and the other of R⁵ and R⁶ is selected fromhydrogen, hydroxy, C₁₋₄alkoxy, and C₁₋₄alkyl, wherein C₁₋₄alkyl isoptionally substituted with halo, wherein each of R¹⁰, R¹¹, R¹², and R¹³is independently hydrogen or C₁₋₄alkyl; or R¹⁰ and R¹¹, together withthe nitrogen atom to which they are attached, or R¹⁰, together with thenitrogen atom to which it is attached and a carbon atom of the adjacentC₁₋₆alkylenyl, or R¹² and R¹³, together with the nitrogen atom to whichthey are attached, or R¹², together with the nitrogen atom to which itis attached and a carbon atom of the adjacent C₁₋₆alkylenyl, form aheterocyclic or heteroaryl ring having from 5 to 7 ring atoms andoptionally containing an additional heteroatom selected from oxygen,nitrogen, and sulfur, wherein nitrogen is optionally substituted with—S(O)₂—C₁₋₄alkyl; and n is 1 or 2; or a pharmaceutically-acceptable saltor stereoisomer thereof.
 13. The method of claim 12 wherein: R⁵ isselected from —O—C₁₋₆alkylenyl-NR¹⁰R¹¹ and C₁₋₆alkylenyl-NR¹²R¹³ and R⁶is hydrogen; or R⁵ is C₁₋₄alkoxy and R⁶ is —C₁₋₆alkylenyl-NR¹²R¹³,wherein each of R¹⁰, R¹¹, R¹² and R¹³ is independently hydrogen orC₁₋₄alkyl, or R¹⁰ and R¹¹, together with the nitrogen atom to which theyare attached, form a piperazinyl ring.
 14. The method of claim 13wherein R⁵ is —O—C₁₋₆alkylenyl-NR¹⁰R¹¹ and R⁶ is hydrogen.
 15. Themethod of claim 12 wherein the compound of formula (II) is selectedfrom: 5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;8-hydroxy-5-[(R)-1-hydroxy-2-(2-{4-[4-(2-piperazin-1-yl-ethoxy)-phenylamino]-phenyl}-ethylamino)-ethyl]-1H-quinolin-2-one;5-[(R)-2-(2-{4-[4-(2-amino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;5[(R)-2-(2-{4-[4-(2-dimethylamino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;and 5[(R)-2-(2-{4-[3-(2-amino-ethyl)-4-methoxy-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;and pharmaceutically-acceptable salts thereof.
 16. The method of claim12 wherein the compound of formula (II) is5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one,or a pharmaceutically-acceptable salt thereof.
 17. The method of claim12 wherein the method comprising administering the compound of formula(II) by inhalation.
 18. The method of claim 12 further comprisingadministering one or more other therapeutic agents wherein the othertherapeutic agent is a corticosteroid or an anticholinergic agent.
 19. Amethod of agonizing a β₂ adrenergic receptor in a mammal, the methodcomprising administering to the mammal a pharmaceutical compositioncomprising a pharmaceutically-acceptable carrier and a compound offormula (II):

wherein: R¹ and R² taken together are —NHC(═O)CH═CH— or —CH═CHC(═O)NH—;one of R⁵ and R⁶ is —[O—C₁₋₆alkylenyl]_(n)-NR¹⁰R¹¹ orC₁₋₆alkylenyl-NR¹²R¹³, and the other of R⁵ and R⁶ is selected fromhydrogen, hydroxy, C₁₋₄alkoxy, and C₁₋₄alkyl, wherein C₁₋₄alkyl isoptionally substituted with halo, wherein each of R¹⁰, R¹¹, R¹², and R¹³is independently hydrogen or C₁₋₄alkyl; or R¹⁰ and R¹¹, together withthe nitrogen atom to which they are attached, or R¹⁰, together with thenitrogen atom to which it is attached and a carbon atom of the adjacentC₁₋₆alkylenyl, or R¹² and R¹³, together with the nitrogen atom to whichthey are attached, or R¹², together with the nitrogen atom to which itis attached and a carbon atom of the adjacent C₁₋₆alkylenyl, form aheterocyclic or heteroaryl ring having from 5 to 7 ring atoms andoptionally containing an additional heteroatom selected from oxygen,nitrogen, and sulfur, wherein nitrogen is optionally substituted with—S(O)₂—C₁₋₄alkyl; and n is 1 or 2; or a pharmaceutically-acceptable saltor stereoisomer thereof.
 20. The method of claim 19 wherein the compoundof formula (II) is selected from: 5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;8-hydroxy-5-[(R)-1-hydroxy-2-(2-{4-[4-(2-piperazin-1-yl-ethoxy)-phenylamino]-phenyl}-ethylamino)-ethyl]-1H-quinolin-2-one;5-[(R)-2-(2-{4-[4-(2-amino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;5-[(R)-2-(2-{4-[4-(2-dimethylamino-ethyl)-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;and 5-[(R)-2-(2-{4-[3-(2-amino-ethyl)-4-methoxy-phenylamino]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one;and pharmaceutically-acceptable salts thereof.